Research Article
Split ViewerAcupoint Autohemotherapy Alleviates Airway Inflammation in Asthmatic Rats via Upregulating Expression of Hemeoxygenase-1
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Hunan Traditional Chinese Medicine College, Zhuzhou, China
Correspondence to:This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
J Acupunct Meridian Stud 2024; 17(5): 149-157
Published October 31, 2024 https://doi.org/10.51507/j.jams.2024.17.5.149
Copyright © Medical Association of Pharmacopuncture Institute.
Abstract
Objective: The role of heme oxygenase-1 (HO-1) in AA-induced suppression of asthmatic airway inflammation is examined.
Methods: Twenty rats were assigned randomly to four groups, namely the Control, OVA, OVA + AA, and (OVA + Snpp) + AA. Rats in the OVA + AA and (OVA + Snpp) + AA received autologous blood injections into acupoints (BL13 and BL23) following OVA challenge. Rats in the (OVA + Snpp) + AA were concurrently subjected to intraperitoneal injections of Snpp, a inhibitor of HO-1. Airway inflammation was evaluated through HE staining, while the concentrations of cytokines in BALF were quantified using ELISA. The mRNA and protein levels of RORγt (Th17-specific transcription factor), Foxp3 (Treg-specific transcription factor), and HO-1 in lung tissue were assessed through qRT-PCR and WB.
Results: HE staining indicated that airway inflammation was alleviated in the OVA + AA. The OVA + AA displayed significantly lower counts of total cells and eosinophils in the BALF compared to both the OVA and (OVA + Snpp) + AA. The ELISA demonstrated a significant decrease in levels of pro-inflamatory cytokines (IL-4, IL-17A), and an increase in levels of anti-inflamatory cytokines (IFN-γ, IL-10), in the OVA + AA when compared to both OVA and (OVA + Snpp) + AA. The qRT-PCR and WB analyses revealed an upregulation of HO-1 and Foxp3 expression, and a downregulation of RORγt expression, in the OVA + AA when compared to OVA and (OVA + Snpp) + AA.
Conclusions and Relevance: The involvement of HO-1 in the underlying mechanism responsible for the anti-inflammatory effects of AA is evident.
Keywords
INTRODUCTION
Bronchial asthma is a chronic inflammatory respiratory disease with steadily increasing global prevalence, affecting human well-being [1]. The condition is characterized by the persistent presence of chronic airway inflammation, causing airway hyperresponsiveness and airway remodeling. Different types of airway inflammatory cells indicate certain inflammatory phenotypes classified into eosinophilic asthma, neutrophilic asthma, mixed granulocytic asthma, and paucigranulocytic asthma [2]. Eosinophilic asthma is the primary clinical asthma phenotype linked to elevated Th2 inflammatory cytokine levels, including interleukin (IL)-4, IL-5, IL-9, and IL-13 [3]. On the other hand, neutrophilic asthma is associated with severe asthma and inflammation mediated by Th17 cells. These cells produce proinflammatory cytokines, such as IL-17A and IL-17F, with IL-17A being more potent than IL-17F [4]. Additionally, Treg cells are implicated in asthma development, with studies demonstrating a reduced number and impaired function of Treg cells in asthma patients [5]. Treg cells exert their function by releasing anti-inflammatory cytokines.
Individuals with asthma frequently exhibit airflow restriction and experience recurrent wheezing, dyspnea, coughing, and chest tightness. While inhaled corticosteroids and β2-agonists are generally effective for patients with asthma, some individuals fail to attain complete symptom control [6]. Acupoint autohemotherapy, involving the subcutaneous injection of autologous blood into acupoints, combines three external therapies derived from Traditional Chinese Medicine: acupuncture, acupoint injection, and bloodletting [7]. Acupoint autohemotherapy is extensively employed in clinical settings for the treatment of diverse diseases, including chronic urticaria [8], acne [9], chronic eczema [10], allergic rhinitis [11], chronic obstructive pulmonary disease [12], and asthma [13]. It has been empirically validated as a secure and efficacious treatment for asthma, capable of mitigating airway inflammation, ameliorating symptoms, and diminishing the frequency of acute attacks [14]. However, the precise mechanism underlying the suppression of inflammation in asthmatic airways by acupoint autohemotherapy remains elusive.
Heme-oxygenase (HO) is the rate-limiting enzyme in heme degradation, metabolizing heme into biliverdin, ferrous iron, and carbon monoxide. Among three HO isozymes (HO-1, HO-2, and HO-3) [15], HO-1 is an inducible isoform that is widely distributed in the microsomes of various tissues in mammalian animals [16]. Various factors, such as endotoxin, hypoxia, inflammatory mediators, cytokines, oxidative stress, and heme and its derivatives, can trigger HO-1 induction [17]. A previous study demonstrated that heme administration through multiple intraperitoneal injections increased HO-1 expression and reduced the infiltration of inflammatory cells in the airways, particularly eosinophils [18]. Given that acupoint autohemotherapy requires repeated autologous blood injections into acupuncture points, we hypothesize that this therapy may induce HO-1 expression, subsequently decreasing the infiltration of inflammatory cells in the airways of rats with asthma. Thus, this study aimed to investigate whether HO-1 induced by acupoint autohemotherapy inhibits airway inflammation in a rat model of asthma using a randomized controlled experimental study design.
METHODS
1. Animals and reagents
Male Sprague Dawley (SD) rats weighing 200-250 g were obtained from the Hubei Provincial Laboratory Animal Center (certificate of conformity: SCXK-2020-0018) and housed under standard conditions with
Ovalbumin (OVA) was purchased from Sigma Co., Ltd (NO. A5503, USA), aluminum hydroxide was purchased from OZ Biosciences Co., Ltd (NO. OZB-AH0050, France), and Tin protoporphyrin IX dichloride (Snpp) was obtained from GLPBIO Co., Ltd (NO. 14325-05-4, USA). Rat IL-4 ELISA kit (NO. E-EL-R0014c), IFN-γ ELISA kit (NO. E-EL-R0009c), IL-17A ELISA kit (NO. E-EL-R0566c), and IL-10 ELISA kit (NO. E-EL-R0016c) were obtained from Elabscience Biotechnology Co., Ltd (China). The study used the following antibodies: rat anti-RORγt (Santa, Sc-293150, USA), rabbit polyclonal antibody to HO-1 (Proteintech, 10701-1-AP, China), rabbit polyclonal antibody to FoxP3 (BIOSS, Bs-0269r, China), horseradish peroxidase (HRP)-labeled sheep anti-rabbit (Beyotime Biotechnology, A0208, China), HRP-labeled sheep anti-mouse (Proteintech, SA00001-1, China), and mouse β-actin (Affinity Biosciences, T0022, USA).
2. Animal grouping and modeling
Twenty SD rats were randomly assigned to four groups (5 rats per group): the control group, the asthma model group (OVA, ovalbumin), the acupoint autohemotherapy group (OVA + AA), and the HO-1 inhibitor group ([OVA + Snpp] + AA). Asthma induction was performed in all rats, except for the control group, using a previously described method [19]. Briefly, the rats received an intraperitoneal injection of 10% OVA complexed with a 10% aluminum hydroxide solution on days 0 and 8, with an injection volume of 1 ml. Subsequently, the rats were intranasally challenged with a 1% OVA solution once daily for 30 minutes each time from day 14 to 28.
Rats belonging to OVA + AA and (OVA + Snpp) + AA groups received autologous blood into acupuncture points, following the procedure outlined in acupoint autohemotherapy, from day 3 to 14 after OVA challenge. Additionally, rats from the (OVA + Snpp) + AA group received intraperitoneal injections of 1% Snpp (an HO-1 inhibitor, 50 μmol/kg) concurrently.
3. Acupoint autohemotherapy
The rats received ocular 2% lidocaine drops to induce surface anesthesia. Subsequently, blood samples (2 ml) were collected from the medial canthus of the eyes using a heparinized capillary tube. The tube was promptly removed, and sterile gauze was applied to the wound for hemostasis. Afterward, ofloxacin eye drops were administered. Subsequently, 200 μl of autologous blood was injected 5 mm below the surface of acupuncture points located at BL13 and BL23.
4. Bronchoalveolar lavage, enzyme-linked immunosorbent assay, and cell counting
The rats were euthanized via exsanguination, followed by trachea isolation and left bronchial tube ligation. Subsequently, a catheter was inserted into the trachea to extract bronchoalveolar lavage fluid (BALF) using a syringe containing 3 ml of phosphate-buffered saline for instillation and aspiration, which was repeated three times. On average, approximately 80% of the BALF was successfully recovered after each lavage procedure. The BALF was preserved in cryotubes and centrifuged at 3,000 rpm for 10 minutes at 4℃. Subsequently, the resulting supernatant was frozen and stored in a low-temperature refrigerator at –80℃ for subsequent cytokine testing. IL-4, interferon (IFN)-γ, IL-17A, and IL-10 concentrations were determined using sandwich enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s guidelines. The remaining sediment was utilized for staining with the Wright-Giemsa solution. The cell numbers were counted on a cell counting plate, differentially classifying cells as macrophages, lymphocytes, eosinophils, and neutrophils.
5. Histological analysis of lung tissue
The left lower lung was isolated and immersed in a 10% buffered formalin solution for 24 hours, followed by paraffin embedment. Subsequently, 4-μm lung sections were stained with hematoxylin and eosin (H&E) and examined using a light microscope to evaluate the presence of airway inflammation.
6. Quantitative real-time polymerase chain reaction
Total RNA was extracted from right lower lung tissue samples using Trizol reagent and subsequently reverse transcribed into cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted with 2x Q3 SYBR qPCR Master Mix (TOLOBIO USA) following the manufacturer’s instructions. The amplification cycle for qRT-PCR consisted of pre-denaturation at 95℃ for 10 minutes, followed by 40 cycles of denaturation at 95℃ for 10 seconds, annealing at 60℃ for 1 minute, and extension at 95℃ for 15 seconds. Table 1 provides the primer sequences for RORγt, FoxP3, HO-1, and β-actin. The relative mRNA level was determined using the 2–ΔΔCt method.
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Table 1 . Primers used for quantitative real-time PCR in this study
Gene Primer Sequence β-actin Forward CACGATGGAGGGGCCGGACTCATC Reverse TAAAGACCTCTATGCCAACACAGT FoxP3 Forward AGGCACTTCTCCAGGACAGA Reverse CTGGACACCCATTCCAGACT HO-1 Forward CACGCATATACCCGCTACCT Reverse AAGGCGGTCTTAGCCTCTTC RORγt Forward GAGGCCATTCAGTACGTGGT Reverse ACACCACCGTATTTGCCTTC
7. Western blotting
The protein extracted from lung tissues was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to separate its components. Subsequently, the separated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. Then, the PVDF membrane was exposed to primary antibodies (RORγt, HO-1, and FoxP3). Following thorough washing, the membrane was incubated with a secondary antibody at room temperature for 2 hours. Finally, the protein bands were visualized using an ECL kit (Servicebio, Wuhan, China), with β-actin serving as the internal reference. The quantification of each band was performed using Image-Pro PLUS software.
8. Statistical analysis
The analysis was performed utilizing the Statistical Package for the Social Sciences (SPSS) version 21.0 (IBM, Chicago, IL, USA). Descriptive statistics, including mean ± standard deviation (SD), were used to describe measurement data with normal distribution. A one-way analysis of variance (ANOVA) was utilized to assess and compare various variables, including the total cell count and eosinophils in BALF, BALF cytokine levels, as well as HO-1, RORγt, and Foxp3 mRNA and protein expression in the rat’s lung tissue across multiple groups. A
RESULTS
1. Acupoint autohemotherapy effects on lung histopathology in rat models of asthma
Fig. 1 demonstrates the histopathological alterations in the lungs identified by H&E staining. Following OVA sensitization and airway challenge, the observed histopathological changes encompassed a substantial infiltration of inflammatory cells in peribronchial and perivascular regions, augmented mucus within the airways, and airway smooth muscle layer thickening in both OVA and (OVA + Snpp) + AA groups. Nevertheless, these aforementioned pathological modifications were comparatively mitigated in the OVA + AA group. Conversely, controls did not exhibit any changes in airway inflammation. The primary complications associated with acupoint autohemotherapy were subcutaneous hematoma or hemorrhage.
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Figure 1.HE staining of lung tissue in rats. There was a significant increase in inflammatory cells (white arrow) around the bronchial and vascular areas, increased mucus in the airways, and thickening of the airway smooth muscle (black arrow) in both the OVA (B) and (OVA + Snpp) + AA (D). However, these changes were less severe in the OVA + AA (C). The images of airways are shown in black boxes. (A) Control (×100); (B) OVA (×100); (C) OVA + AA (×100); (D) (OVA + Snpp) + AA (×100). OVA = ovalbumin; AA = acupoint autohemotherapy; Snpp = Tin protoporphyrin IX dichloride, a HO-1 inhibitor.
2. Acupoint autohemotherapy effects on BALF cellular composition in rat models of asthma
According to the findings presented in Fig. 2, the rats in the OVA groups exhibited a notably higher count of total cells (
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Figure 2.Cell counts in BALF of rats. (A) Total cell count in BALF (×107). (B) Percentage of eosinophils (EOS) in BALF (%). BALF = bronchoalveolar lavage fluid; OVA = ovalbumin; AA = acupoint autohemotherapy; Snpp = Tin protoporphyrin IX dichloride, a HO-1 inhibitor. All the data are presented as the means ± SD. aCompared with Control,
p < 0.05; bcompared with OVA,p < 0.05; ccompared with OVA + AA,p < 0.05; dcompared with (OVA + Snpp) + AA,p < 0.05.
3. Acupoint autohemotherapy effects on BALF cytokine levels in rat models with asthma
Previous research demonstrated that an imbalance in T lymphocyte subsets, specifically Th1/Th2 and Treg/Th17, plays a role in asthma development. Thus, we conducted ELISA to measure the levels of cytokines associated with Th1, Th2, Treg, and Th17 for further investigation. Compared to controls, the OVA group exhibited significantly elevated IL-4 (
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Figure 3.Cytokines levels in BALF of rats. (A) Levels of Th2-related cytokine (IL-4). (B) Levels of Th1-related cytokine (IFN-γ). (C) Levels of Th17-related cytokine (IL-17A). (D) Levels of Treg-related cytokine (IL-10). BALF = bronchoalveolar lavage fluid; OVA = ovalbumin; AA = acupoint autohemotherapy; Snpp = Tin protoporphyrin IX dichloride, a HO-1 inhibitor. All the data are presented as the means ± SD. aCompared with Control,
p < 0.05; bcompared with OVA,p < 0.05; ccompared with OVA + AA,p < 0.05; dcompared with (OVA + Snpp) + AA,p < 0.05.
4. Acupoint autohemotherapy effects on HO-1 mRNA and protein expression in the lung tissue in rat models of asthma
HO-1 mRNA and protein levels in the lung tissue were assessed using qRT-PCR and western blot, respectively. The OVA group exhibited significantly elevated HO-1 mRNA (
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Table 2 . Quantitative real-time PCR results
Group n HO-1 RORγt FoxP3 Control 5 1.005 ± 0.144 1.039 ± 0.103 1.041 ± 0.135 OVA 5 1.533 ± 0.144*,‡ 3.017 ± 0.300*,‡,§ 0.353 ± 0.035 *,‡ OVA + AA 5 3.031 ± 0.335*,†,§ 1.436 ± 0.174†,§ 0.855 ± 0.087*,†,§ (OVA + Snpp) + AA 5 1.721 ± 0.196*,‡ 2.268 ± 0.254*,†,‡ 0.488 ± 0.047*,‡ All the data are presented as the means ± SD. *Compared with Control,
p < 0.05; †compared with OVA,p < 0.05; ‡compared with OVA + AA,p < 0.05; §compared with (OVA + Snpp) + AA,p < 0.05.
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Figure 4.Western blotting results (A) and gray analysis of western blotting (B-D). (B) HO-1 protein normalized to β-actin. (C) RORγt protein normalized to β-actin. (D) Foxp3 protein normalized to β-actin. OVA = ovalbumin; AA = acupoint autohemotherapy; Snpp = Tin protoporphyrin IX dichloride, a HO-1 inhibitor. All the data are presented as the means ± SD. aCompared with Control,
p < 0.05; bcompared with OVA,p < 0.05; ccompared with OVA + AA,p < 0.05; dcompared with (OVA + Snpp) + AA,p < 0.05.
5. Acupoint autohemotherapy effects on Foxp3 and RORγt mRNA and protein expression in the lung tissue in rat models of asthma
Considering that the regulation of CD4+ T cell differentiation into Treg or Th17 cells primarily relies on nuclear transcription factors, we employed qRT-PCR and western blot to measure Foxp3 and RORγt mRNA and protein levels. The findings depicted in Table 2 and Fig. 4 demonstrate that the OVA group exhibited significantly lower Foxp3 mRNA (
DISCUSSION
In this study, a rat model of asthma was developed to examine acupoint autohemotherapy effects on airway inflammation. Acupoint autohemotherapy could mitigate the infiltration of airway inflammatory cells, promote the secretion of anti-inflammatory Th1 and Treg cytokines, and reduce the production of pro-inflammatory Th2 and Th17 cytokines. Therefore, acupoint autohemotherapy may be an effective intervention for attenuating airway inflammation in experimental asthma.
Research showed that acupoint autohemotherapy may mimic hematoma absorption in the muscular tissue, with local inflammation being initiated to engulf and clear the blood components [20]. Hemin, which acts as an HO-1 substrate, is a physiological inducer of HO-1 that increases both HO-1 mRNA and protein levels [18]. The present study revealed that HO-1 expression in the lung tissues in the OVA + AA group was markedly higher than in the OVA group. Additionally, the numbers of eosinophils and total inflammatory cells in BALF were significantly reduced in the OVA + AA group compared to the OVA group. Hence, the anti-inflammatory effects of acupoint autohemotherapy were associated with HO-1 induction. However, HO-1 expression was markedly decreased following treatment with HO-1 inhibitor Snpp. Moreover, the presence of airway inflammation and BALF eosinophil count in the (OVA + Snpp) + AA group were comparable to those in the OVA group. Thus, the HO-1 inhibitor could offset the protective effects of acupoint autohemotherapy on airway inflammation in rats with asthma.
HO-1 possesses cytoprotective properties, encompassing antioxidant, antiapoptotic, and anti-inflammatory activities [17]. HO-1 could be induced in response to stress, such as oxidative stress, hypoxia, and inflammatory conditions [16]. This study showed increased HO-1 expression in the lung tissues in the OVA group compared to controls. This phenomenon may be ascribed to the induction of HO-1 expression by airway inflammation. However, the cytoprotective efficacy conferred by the upregulated HO-1 in rats with asthma proved insufficient in countering inflammation-induced damage [17]. Hence, a greater increase in HO-1 is necessary to effectively suppress airway inflammation in rats with asthma. Therefore, this study demonstrates that acupoint autohemotherapy can meet these requirements.
Numerous studies demonstrated the significance of Th1/Th2 immune imbalance in asthma development, characterized by an augmented Th2 immune response and diminished Th1 immune response [21]. In this study, we observed a substantially reduced IL-4 concentration in BALF in the OVA + AA group and significantly increased IFN-γ, a Th1 cytokine, compared to the OVA group. Interestingly, HO-1 inhibitor Snpp reversed this effect. Thus, acupoint autohemotherapy could facilitate the release of Th1 cytokines while suppressing the production of Th2 cytokines, thereby modulating the Th1/Th2 balance by increasing HO-1 expression.
In addition to the Th1/Th2 imbalance, the dysregulation of Th17/Treg is also implicated in the etiology of asthma [21]. Treg cells, crucial immune-suppressive cells, play a pivotal role in immune tolerance and inflammation suppression by secreting cytokines such as IL-10 and transforming growth factor (TGF)-β. Conversely, pro-inflammatory Th17 cells generate IL-17A, a pivotal mediator of neutrophilic inflammation. Treg and Th17 cells play a crucial role in maintaining immune homeostasis by engaging in the reciprocal regulation of differentiation and function [22]. Regarding asthma pathogenesis, a reduced Treg/Th17 ratio is evident, accompanied by decreased Treg-related cytokine levels and increased Th17-related cytokine levels [23]. Our experimental findings corroborate this observation. Additionally, our results demonstrated that acupoint autohemotherapy could attenuate IL-17A secretion and increase IL-10 secretion in the BALF of rats with asthma. Thus, acupoint autohemotherapy may partially restore the equilibrium between Treg and Th17 cells. The potential mechanism underlying this phenomenon might be associated with the regulation of T cell transcription factors, specifically FoxP3 and RORγt [24]. We found that the OVA + AA group exhibited higher FoxP3 expression and lower RORγt expression in the lung tissue compared to the OVA group. Previous research suggested that the balance between Treg and Th17 cells, which is implicated in asthma-related inflammation, could potentially be mediated by the ratio of FoxP3 to RORγt [24]. In our experiment, the HO-1 inhibitor effectively reversed the effect of acupoint autohemotherapy on the Treg/Th17 balance. Therefore, acupoint autohemotherapy modulates the Treg/Th17 balance by upregulating FoxP3 expression and downregulating RORγt expression levels through HO-1.
In addition to the pharmacological action of autologous blood, acupoint autohemotherapy exhibits a protective effect on asthma. This study focused on two specific acupoints, namely BL13 and BL23, which are recognized as the Back-Shu points of the Foot Taiyang bladder meridian and are commonly employed in asthma treatment by acupuncture. According to Traditional Chinese Medicine theories, BL13 possesses a substantial amount of Qi and blood associated with the lung, enabling it to dispel wind, relieve the exterior, and regulate lung Qi. Research demonstrated that acupuncture treatment targeting the BL13 acupoint is associated with enhanced lung function, including improvements in the forced vital capacity and forced expiratory volume, as well as the reduction in small airway resistance [25]. The BL23 acupoint stimulation enhances inspiration by fortifying the kidney. Furthermore, investigations revealed significantly elevated cortisol levels following acupuncture at the BL23 acupoint [26]. Mo et al. [13] revealed that saline injections in the BL13 and BL23 acupoints of rats with asthma partially ameliorated the Th1/Th2 imbalance, although it did not significantly influence the Th17/Treg imbalance.
CONCLUSIONS
Thus, acupoint autohemotherapy effectively corrects both the Th1/Th2 imbalance and Th17/Treg imbalance in rats with asthma, indicating that autologous blood plays a crucial role in asthma treatment through acupoint autohemotherapy. The present study also provided evidence suggesting that upregulated HO-1 expression may be involved in the mechanism responsible for the anti-inflammatory effect of acupoint autohemotherapy.
This work has several limitations. One is the absence of a control group receiving an alternative injection, such as normal saline, to facilitate a comparative analysis of the effects of autologous blood and acupuncture point stimulation. The attainment of “De qi” is crucial for the desired therapeutic outcomes of acupuncture therapy. “De qi” is characterized as a sensation of warmth, tightening, or deep soreness. However, the assessment of these subjective sensations in experimental animals poses significant challenges. Another limitation is the exploratory nature of the study, limiting the sample size. Consequently, an augmented sample size is required to fortify the findings of this study.
FUNDING
This research was support by the Research Project of Hunan Provincial Health Commission (No. D202303027756).
AUTHORS’ CONTRIBUTIONS
Conceived and designed the experiments: Shi-kui Wu, Hao-lei Liu; Performed the experiments: Tao Wu, Hao-lei Liu. Analyzed the data: Xiang Zeng; Wrote the paper: Hao-lei Liu; Supervised the research: Wei-yun Cao, Shi-kui Wu; All authors have read and agreed to the published version of the manuscript.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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Article
Research Article
J Acupunct Meridian Stud 2024; 17(5): 149-157
Published online October 31, 2024 https://doi.org/10.51507/j.jams.2024.17.5.149
Copyright © Medical Association of Pharmacopuncture Institute.
Acupoint Autohemotherapy Alleviates Airway Inflammation in Asthmatic Rats via Upregulating Expression of Hemeoxygenase-1
Hao-lei Liu* , Tao Wu, Xiang Zeng, Wei-yun Cao, Shi-kui Wu
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Hunan Traditional Chinese Medicine College, Zhuzhou, China
Correspondence to:Hao-lei Liu
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Hunan Traditional Chinese Medicine College, Zhuzhou, China
E-mail howlate555@163.com
This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Importance: Acupoint autohemotherapy (AA), a therapeutic technique involving the subcutaneous injection of autologous blood into acupoints, has been empirically validated as safe and effective for treating asthma by alleviating symptoms and decreasing acute attacks, though its mechanism is not well understood.
Objective: The role of heme oxygenase-1 (HO-1) in AA-induced suppression of asthmatic airway inflammation is examined.
Methods: Twenty rats were assigned randomly to four groups, namely the Control, OVA, OVA + AA, and (OVA + Snpp) + AA. Rats in the OVA + AA and (OVA + Snpp) + AA received autologous blood injections into acupoints (BL13 and BL23) following OVA challenge. Rats in the (OVA + Snpp) + AA were concurrently subjected to intraperitoneal injections of Snpp, a inhibitor of HO-1. Airway inflammation was evaluated through HE staining, while the concentrations of cytokines in BALF were quantified using ELISA. The mRNA and protein levels of RORγt (Th17-specific transcription factor), Foxp3 (Treg-specific transcription factor), and HO-1 in lung tissue were assessed through qRT-PCR and WB.
Results: HE staining indicated that airway inflammation was alleviated in the OVA + AA. The OVA + AA displayed significantly lower counts of total cells and eosinophils in the BALF compared to both the OVA and (OVA + Snpp) + AA. The ELISA demonstrated a significant decrease in levels of pro-inflamatory cytokines (IL-4, IL-17A), and an increase in levels of anti-inflamatory cytokines (IFN-γ, IL-10), in the OVA + AA when compared to both OVA and (OVA + Snpp) + AA. The qRT-PCR and WB analyses revealed an upregulation of HO-1 and Foxp3 expression, and a downregulation of RORγt expression, in the OVA + AA when compared to OVA and (OVA + Snpp) + AA.
Conclusions and Relevance: The involvement of HO-1 in the underlying mechanism responsible for the anti-inflammatory effects of AA is evident.
Keywords: Acupoint autohemotherapy, Asthma, Heme oxygenase-1, Airway inflammation, Autologous blood, Cytokines
INTRODUCTION
Bronchial asthma is a chronic inflammatory respiratory disease with steadily increasing global prevalence, affecting human well-being [1]. The condition is characterized by the persistent presence of chronic airway inflammation, causing airway hyperresponsiveness and airway remodeling. Different types of airway inflammatory cells indicate certain inflammatory phenotypes classified into eosinophilic asthma, neutrophilic asthma, mixed granulocytic asthma, and paucigranulocytic asthma [2]. Eosinophilic asthma is the primary clinical asthma phenotype linked to elevated Th2 inflammatory cytokine levels, including interleukin (IL)-4, IL-5, IL-9, and IL-13 [3]. On the other hand, neutrophilic asthma is associated with severe asthma and inflammation mediated by Th17 cells. These cells produce proinflammatory cytokines, such as IL-17A and IL-17F, with IL-17A being more potent than IL-17F [4]. Additionally, Treg cells are implicated in asthma development, with studies demonstrating a reduced number and impaired function of Treg cells in asthma patients [5]. Treg cells exert their function by releasing anti-inflammatory cytokines.
Individuals with asthma frequently exhibit airflow restriction and experience recurrent wheezing, dyspnea, coughing, and chest tightness. While inhaled corticosteroids and β2-agonists are generally effective for patients with asthma, some individuals fail to attain complete symptom control [6]. Acupoint autohemotherapy, involving the subcutaneous injection of autologous blood into acupoints, combines three external therapies derived from Traditional Chinese Medicine: acupuncture, acupoint injection, and bloodletting [7]. Acupoint autohemotherapy is extensively employed in clinical settings for the treatment of diverse diseases, including chronic urticaria [8], acne [9], chronic eczema [10], allergic rhinitis [11], chronic obstructive pulmonary disease [12], and asthma [13]. It has been empirically validated as a secure and efficacious treatment for asthma, capable of mitigating airway inflammation, ameliorating symptoms, and diminishing the frequency of acute attacks [14]. However, the precise mechanism underlying the suppression of inflammation in asthmatic airways by acupoint autohemotherapy remains elusive.
Heme-oxygenase (HO) is the rate-limiting enzyme in heme degradation, metabolizing heme into biliverdin, ferrous iron, and carbon monoxide. Among three HO isozymes (HO-1, HO-2, and HO-3) [15], HO-1 is an inducible isoform that is widely distributed in the microsomes of various tissues in mammalian animals [16]. Various factors, such as endotoxin, hypoxia, inflammatory mediators, cytokines, oxidative stress, and heme and its derivatives, can trigger HO-1 induction [17]. A previous study demonstrated that heme administration through multiple intraperitoneal injections increased HO-1 expression and reduced the infiltration of inflammatory cells in the airways, particularly eosinophils [18]. Given that acupoint autohemotherapy requires repeated autologous blood injections into acupuncture points, we hypothesize that this therapy may induce HO-1 expression, subsequently decreasing the infiltration of inflammatory cells in the airways of rats with asthma. Thus, this study aimed to investigate whether HO-1 induced by acupoint autohemotherapy inhibits airway inflammation in a rat model of asthma using a randomized controlled experimental study design.
METHODS
1. Animals and reagents
Male Sprague Dawley (SD) rats weighing 200-250 g were obtained from the Hubei Provincial Laboratory Animal Center (certificate of conformity: SCXK-2020-0018) and housed under standard conditions with
Ovalbumin (OVA) was purchased from Sigma Co., Ltd (NO. A5503, USA), aluminum hydroxide was purchased from OZ Biosciences Co., Ltd (NO. OZB-AH0050, France), and Tin protoporphyrin IX dichloride (Snpp) was obtained from GLPBIO Co., Ltd (NO. 14325-05-4, USA). Rat IL-4 ELISA kit (NO. E-EL-R0014c), IFN-γ ELISA kit (NO. E-EL-R0009c), IL-17A ELISA kit (NO. E-EL-R0566c), and IL-10 ELISA kit (NO. E-EL-R0016c) were obtained from Elabscience Biotechnology Co., Ltd (China). The study used the following antibodies: rat anti-RORγt (Santa, Sc-293150, USA), rabbit polyclonal antibody to HO-1 (Proteintech, 10701-1-AP, China), rabbit polyclonal antibody to FoxP3 (BIOSS, Bs-0269r, China), horseradish peroxidase (HRP)-labeled sheep anti-rabbit (Beyotime Biotechnology, A0208, China), HRP-labeled sheep anti-mouse (Proteintech, SA00001-1, China), and mouse β-actin (Affinity Biosciences, T0022, USA).
2. Animal grouping and modeling
Twenty SD rats were randomly assigned to four groups (5 rats per group): the control group, the asthma model group (OVA, ovalbumin), the acupoint autohemotherapy group (OVA + AA), and the HO-1 inhibitor group ([OVA + Snpp] + AA). Asthma induction was performed in all rats, except for the control group, using a previously described method [19]. Briefly, the rats received an intraperitoneal injection of 10% OVA complexed with a 10% aluminum hydroxide solution on days 0 and 8, with an injection volume of 1 ml. Subsequently, the rats were intranasally challenged with a 1% OVA solution once daily for 30 minutes each time from day 14 to 28.
Rats belonging to OVA + AA and (OVA + Snpp) + AA groups received autologous blood into acupuncture points, following the procedure outlined in acupoint autohemotherapy, from day 3 to 14 after OVA challenge. Additionally, rats from the (OVA + Snpp) + AA group received intraperitoneal injections of 1% Snpp (an HO-1 inhibitor, 50 μmol/kg) concurrently.
3. Acupoint autohemotherapy
The rats received ocular 2% lidocaine drops to induce surface anesthesia. Subsequently, blood samples (2 ml) were collected from the medial canthus of the eyes using a heparinized capillary tube. The tube was promptly removed, and sterile gauze was applied to the wound for hemostasis. Afterward, ofloxacin eye drops were administered. Subsequently, 200 μl of autologous blood was injected 5 mm below the surface of acupuncture points located at BL13 and BL23.
4. Bronchoalveolar lavage, enzyme-linked immunosorbent assay, and cell counting
The rats were euthanized via exsanguination, followed by trachea isolation and left bronchial tube ligation. Subsequently, a catheter was inserted into the trachea to extract bronchoalveolar lavage fluid (BALF) using a syringe containing 3 ml of phosphate-buffered saline for instillation and aspiration, which was repeated three times. On average, approximately 80% of the BALF was successfully recovered after each lavage procedure. The BALF was preserved in cryotubes and centrifuged at 3,000 rpm for 10 minutes at 4℃. Subsequently, the resulting supernatant was frozen and stored in a low-temperature refrigerator at –80℃ for subsequent cytokine testing. IL-4, interferon (IFN)-γ, IL-17A, and IL-10 concentrations were determined using sandwich enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s guidelines. The remaining sediment was utilized for staining with the Wright-Giemsa solution. The cell numbers were counted on a cell counting plate, differentially classifying cells as macrophages, lymphocytes, eosinophils, and neutrophils.
5. Histological analysis of lung tissue
The left lower lung was isolated and immersed in a 10% buffered formalin solution for 24 hours, followed by paraffin embedment. Subsequently, 4-μm lung sections were stained with hematoxylin and eosin (H&E) and examined using a light microscope to evaluate the presence of airway inflammation.
6. Quantitative real-time polymerase chain reaction
Total RNA was extracted from right lower lung tissue samples using Trizol reagent and subsequently reverse transcribed into cDNA. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted with 2x Q3 SYBR qPCR Master Mix (TOLOBIO USA) following the manufacturer’s instructions. The amplification cycle for qRT-PCR consisted of pre-denaturation at 95℃ for 10 minutes, followed by 40 cycles of denaturation at 95℃ for 10 seconds, annealing at 60℃ for 1 minute, and extension at 95℃ for 15 seconds. Table 1 provides the primer sequences for RORγt, FoxP3, HO-1, and β-actin. The relative mRNA level was determined using the 2–ΔΔCt method.
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Table 1
Primers used for quantitative real-time PCR in this study.
Gene Primer Sequence β-actin Forward CACGATGGAGGGGCCGGACTCATC Reverse TAAAGACCTCTATGCCAACACAGT FoxP3 Forward AGGCACTTCTCCAGGACAGA Reverse CTGGACACCCATTCCAGACT HO-1 Forward CACGCATATACCCGCTACCT Reverse AAGGCGGTCTTAGCCTCTTC RORγt Forward GAGGCCATTCAGTACGTGGT Reverse ACACCACCGTATTTGCCTTC
7. Western blotting
The protein extracted from lung tissues was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) to separate its components. Subsequently, the separated proteins were transferred onto a polyvinylidene fluoride (PVDF) membrane. Then, the PVDF membrane was exposed to primary antibodies (RORγt, HO-1, and FoxP3). Following thorough washing, the membrane was incubated with a secondary antibody at room temperature for 2 hours. Finally, the protein bands were visualized using an ECL kit (Servicebio, Wuhan, China), with β-actin serving as the internal reference. The quantification of each band was performed using Image-Pro PLUS software.
8. Statistical analysis
The analysis was performed utilizing the Statistical Package for the Social Sciences (SPSS) version 21.0 (IBM, Chicago, IL, USA). Descriptive statistics, including mean ± standard deviation (SD), were used to describe measurement data with normal distribution. A one-way analysis of variance (ANOVA) was utilized to assess and compare various variables, including the total cell count and eosinophils in BALF, BALF cytokine levels, as well as HO-1, RORγt, and Foxp3 mRNA and protein expression in the rat’s lung tissue across multiple groups. A
RESULTS
1. Acupoint autohemotherapy effects on lung histopathology in rat models of asthma
Fig. 1 demonstrates the histopathological alterations in the lungs identified by H&E staining. Following OVA sensitization and airway challenge, the observed histopathological changes encompassed a substantial infiltration of inflammatory cells in peribronchial and perivascular regions, augmented mucus within the airways, and airway smooth muscle layer thickening in both OVA and (OVA + Snpp) + AA groups. Nevertheless, these aforementioned pathological modifications were comparatively mitigated in the OVA + AA group. Conversely, controls did not exhibit any changes in airway inflammation. The primary complications associated with acupoint autohemotherapy were subcutaneous hematoma or hemorrhage.
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Figure 1. HE staining of lung tissue in rats. There was a significant increase in inflammatory cells (white arrow) around the bronchial and vascular areas, increased mucus in the airways, and thickening of the airway smooth muscle (black arrow) in both the OVA (B) and (OVA + Snpp) + AA (D). However, these changes were less severe in the OVA + AA (C). The images of airways are shown in black boxes. (A) Control (×100); (B) OVA (×100); (C) OVA + AA (×100); (D) (OVA + Snpp) + AA (×100). OVA = ovalbumin; AA = acupoint autohemotherapy; Snpp = Tin protoporphyrin IX dichloride, a HO-1 inhibitor.
2. Acupoint autohemotherapy effects on BALF cellular composition in rat models of asthma
According to the findings presented in Fig. 2, the rats in the OVA groups exhibited a notably higher count of total cells (
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Figure 2. Cell counts in BALF of rats. (A) Total cell count in BALF (×107). (B) Percentage of eosinophils (EOS) in BALF (%). BALF = bronchoalveolar lavage fluid; OVA = ovalbumin; AA = acupoint autohemotherapy; Snpp = Tin protoporphyrin IX dichloride, a HO-1 inhibitor. All the data are presented as the means ± SD. aCompared with Control,
p < 0.05; bcompared with OVA,p < 0.05; ccompared with OVA + AA,p < 0.05; dcompared with (OVA + Snpp) + AA,p < 0.05.
3. Acupoint autohemotherapy effects on BALF cytokine levels in rat models with asthma
Previous research demonstrated that an imbalance in T lymphocyte subsets, specifically Th1/Th2 and Treg/Th17, plays a role in asthma development. Thus, we conducted ELISA to measure the levels of cytokines associated with Th1, Th2, Treg, and Th17 for further investigation. Compared to controls, the OVA group exhibited significantly elevated IL-4 (
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Figure 3. Cytokines levels in BALF of rats. (A) Levels of Th2-related cytokine (IL-4). (B) Levels of Th1-related cytokine (IFN-γ). (C) Levels of Th17-related cytokine (IL-17A). (D) Levels of Treg-related cytokine (IL-10). BALF = bronchoalveolar lavage fluid; OVA = ovalbumin; AA = acupoint autohemotherapy; Snpp = Tin protoporphyrin IX dichloride, a HO-1 inhibitor. All the data are presented as the means ± SD. aCompared with Control,
p < 0.05; bcompared with OVA,p < 0.05; ccompared with OVA + AA,p < 0.05; dcompared with (OVA + Snpp) + AA,p < 0.05.
4. Acupoint autohemotherapy effects on HO-1 mRNA and protein expression in the lung tissue in rat models of asthma
HO-1 mRNA and protein levels in the lung tissue were assessed using qRT-PCR and western blot, respectively. The OVA group exhibited significantly elevated HO-1 mRNA (
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&md=tbl&idx=2' data-target="#file-modal"">Table 2
Quantitative real-time PCR results.
Group n HO-1 RORγt FoxP3 Control 5 1.005 ± 0.144 1.039 ± 0.103 1.041 ± 0.135 OVA 5 1.533 ± 0.144*,‡ 3.017 ± 0.300*,‡,§ 0.353 ± 0.035 *,‡ OVA + AA 5 3.031 ± 0.335*,†,§ 1.436 ± 0.174†,§ 0.855 ± 0.087*,†,§ (OVA + Snpp) + AA 5 1.721 ± 0.196*,‡ 2.268 ± 0.254*,†,‡ 0.488 ± 0.047*,‡ All the data are presented as the means ± SD. *Compared with Control,
p < 0.05; †compared with OVA,p < 0.05; ‡compared with OVA + AA,p < 0.05; §compared with (OVA + Snpp) + AA,p < 0.05..
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Figure 4. Western blotting results (A) and gray analysis of western blotting (B-D). (B) HO-1 protein normalized to β-actin. (C) RORγt protein normalized to β-actin. (D) Foxp3 protein normalized to β-actin. OVA = ovalbumin; AA = acupoint autohemotherapy; Snpp = Tin protoporphyrin IX dichloride, a HO-1 inhibitor. All the data are presented as the means ± SD. aCompared with Control,
p < 0.05; bcompared with OVA,p < 0.05; ccompared with OVA + AA,p < 0.05; dcompared with (OVA + Snpp) + AA,p < 0.05.
5. Acupoint autohemotherapy effects on Foxp3 and RORγt mRNA and protein expression in the lung tissue in rat models of asthma
Considering that the regulation of CD4+ T cell differentiation into Treg or Th17 cells primarily relies on nuclear transcription factors, we employed qRT-PCR and western blot to measure Foxp3 and RORγt mRNA and protein levels. The findings depicted in Table 2 and Fig. 4 demonstrate that the OVA group exhibited significantly lower Foxp3 mRNA (
DISCUSSION
In this study, a rat model of asthma was developed to examine acupoint autohemotherapy effects on airway inflammation. Acupoint autohemotherapy could mitigate the infiltration of airway inflammatory cells, promote the secretion of anti-inflammatory Th1 and Treg cytokines, and reduce the production of pro-inflammatory Th2 and Th17 cytokines. Therefore, acupoint autohemotherapy may be an effective intervention for attenuating airway inflammation in experimental asthma.
Research showed that acupoint autohemotherapy may mimic hematoma absorption in the muscular tissue, with local inflammation being initiated to engulf and clear the blood components [20]. Hemin, which acts as an HO-1 substrate, is a physiological inducer of HO-1 that increases both HO-1 mRNA and protein levels [18]. The present study revealed that HO-1 expression in the lung tissues in the OVA + AA group was markedly higher than in the OVA group. Additionally, the numbers of eosinophils and total inflammatory cells in BALF were significantly reduced in the OVA + AA group compared to the OVA group. Hence, the anti-inflammatory effects of acupoint autohemotherapy were associated with HO-1 induction. However, HO-1 expression was markedly decreased following treatment with HO-1 inhibitor Snpp. Moreover, the presence of airway inflammation and BALF eosinophil count in the (OVA + Snpp) + AA group were comparable to those in the OVA group. Thus, the HO-1 inhibitor could offset the protective effects of acupoint autohemotherapy on airway inflammation in rats with asthma.
HO-1 possesses cytoprotective properties, encompassing antioxidant, antiapoptotic, and anti-inflammatory activities [17]. HO-1 could be induced in response to stress, such as oxidative stress, hypoxia, and inflammatory conditions [16]. This study showed increased HO-1 expression in the lung tissues in the OVA group compared to controls. This phenomenon may be ascribed to the induction of HO-1 expression by airway inflammation. However, the cytoprotective efficacy conferred by the upregulated HO-1 in rats with asthma proved insufficient in countering inflammation-induced damage [17]. Hence, a greater increase in HO-1 is necessary to effectively suppress airway inflammation in rats with asthma. Therefore, this study demonstrates that acupoint autohemotherapy can meet these requirements.
Numerous studies demonstrated the significance of Th1/Th2 immune imbalance in asthma development, characterized by an augmented Th2 immune response and diminished Th1 immune response [21]. In this study, we observed a substantially reduced IL-4 concentration in BALF in the OVA + AA group and significantly increased IFN-γ, a Th1 cytokine, compared to the OVA group. Interestingly, HO-1 inhibitor Snpp reversed this effect. Thus, acupoint autohemotherapy could facilitate the release of Th1 cytokines while suppressing the production of Th2 cytokines, thereby modulating the Th1/Th2 balance by increasing HO-1 expression.
In addition to the Th1/Th2 imbalance, the dysregulation of Th17/Treg is also implicated in the etiology of asthma [21]. Treg cells, crucial immune-suppressive cells, play a pivotal role in immune tolerance and inflammation suppression by secreting cytokines such as IL-10 and transforming growth factor (TGF)-β. Conversely, pro-inflammatory Th17 cells generate IL-17A, a pivotal mediator of neutrophilic inflammation. Treg and Th17 cells play a crucial role in maintaining immune homeostasis by engaging in the reciprocal regulation of differentiation and function [22]. Regarding asthma pathogenesis, a reduced Treg/Th17 ratio is evident, accompanied by decreased Treg-related cytokine levels and increased Th17-related cytokine levels [23]. Our experimental findings corroborate this observation. Additionally, our results demonstrated that acupoint autohemotherapy could attenuate IL-17A secretion and increase IL-10 secretion in the BALF of rats with asthma. Thus, acupoint autohemotherapy may partially restore the equilibrium between Treg and Th17 cells. The potential mechanism underlying this phenomenon might be associated with the regulation of T cell transcription factors, specifically FoxP3 and RORγt [24]. We found that the OVA + AA group exhibited higher FoxP3 expression and lower RORγt expression in the lung tissue compared to the OVA group. Previous research suggested that the balance between Treg and Th17 cells, which is implicated in asthma-related inflammation, could potentially be mediated by the ratio of FoxP3 to RORγt [24]. In our experiment, the HO-1 inhibitor effectively reversed the effect of acupoint autohemotherapy on the Treg/Th17 balance. Therefore, acupoint autohemotherapy modulates the Treg/Th17 balance by upregulating FoxP3 expression and downregulating RORγt expression levels through HO-1.
In addition to the pharmacological action of autologous blood, acupoint autohemotherapy exhibits a protective effect on asthma. This study focused on two specific acupoints, namely BL13 and BL23, which are recognized as the Back-Shu points of the Foot Taiyang bladder meridian and are commonly employed in asthma treatment by acupuncture. According to Traditional Chinese Medicine theories, BL13 possesses a substantial amount of Qi and blood associated with the lung, enabling it to dispel wind, relieve the exterior, and regulate lung Qi. Research demonstrated that acupuncture treatment targeting the BL13 acupoint is associated with enhanced lung function, including improvements in the forced vital capacity and forced expiratory volume, as well as the reduction in small airway resistance [25]. The BL23 acupoint stimulation enhances inspiration by fortifying the kidney. Furthermore, investigations revealed significantly elevated cortisol levels following acupuncture at the BL23 acupoint [26]. Mo et al. [13] revealed that saline injections in the BL13 and BL23 acupoints of rats with asthma partially ameliorated the Th1/Th2 imbalance, although it did not significantly influence the Th17/Treg imbalance.
CONCLUSIONS
Thus, acupoint autohemotherapy effectively corrects both the Th1/Th2 imbalance and Th17/Treg imbalance in rats with asthma, indicating that autologous blood plays a crucial role in asthma treatment through acupoint autohemotherapy. The present study also provided evidence suggesting that upregulated HO-1 expression may be involved in the mechanism responsible for the anti-inflammatory effect of acupoint autohemotherapy.
This work has several limitations. One is the absence of a control group receiving an alternative injection, such as normal saline, to facilitate a comparative analysis of the effects of autologous blood and acupuncture point stimulation. The attainment of “De qi” is crucial for the desired therapeutic outcomes of acupuncture therapy. “De qi” is characterized as a sensation of warmth, tightening, or deep soreness. However, the assessment of these subjective sensations in experimental animals poses significant challenges. Another limitation is the exploratory nature of the study, limiting the sample size. Consequently, an augmented sample size is required to fortify the findings of this study.
FUNDING
This research was support by the Research Project of Hunan Provincial Health Commission (No. D202303027756).
AUTHORS’ CONTRIBUTIONS
Conceived and designed the experiments: Shi-kui Wu, Hao-lei Liu; Performed the experiments: Tao Wu, Hao-lei Liu. Analyzed the data: Xiang Zeng; Wrote the paper: Hao-lei Liu; Supervised the research: Wei-yun Cao, Shi-kui Wu; All authors have read and agreed to the published version of the manuscript.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
Fig 1.
Fig 2.
Fig 3.
Fig 4.
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Table 1 . Primers used for quantitative real-time PCR in this study.
Gene Primer Sequence β-actin Forward CACGATGGAGGGGCCGGACTCATC Reverse TAAAGACCTCTATGCCAACACAGT FoxP3 Forward AGGCACTTCTCCAGGACAGA Reverse CTGGACACCCATTCCAGACT HO-1 Forward CACGCATATACCCGCTACCT Reverse AAGGCGGTCTTAGCCTCTTC RORγt Forward GAGGCCATTCAGTACGTGGT Reverse ACACCACCGTATTTGCCTTC
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Table 2 . Quantitative real-time PCR results.
Group n HO-1 RORγt FoxP3 Control 5 1.005 ± 0.144 1.039 ± 0.103 1.041 ± 0.135 OVA 5 1.533 ± 0.144*,‡ 3.017 ± 0.300*,‡,§ 0.353 ± 0.035 *,‡ OVA + AA 5 3.031 ± 0.335*,†,§ 1.436 ± 0.174†,§ 0.855 ± 0.087*,†,§ (OVA + Snpp) + AA 5 1.721 ± 0.196*,‡ 2.268 ± 0.254*,†,‡ 0.488 ± 0.047*,‡ All the data are presented as the means ± SD. *Compared with Control,
p < 0.05; †compared with OVA,p < 0.05; ‡compared with OVA + AA,p < 0.05; §compared with (OVA + Snpp) + AA,p < 0.05..
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