“肺俞”“天枢”艾灸对支气管哮喘模型大鼠气道重塑、免疫细胞分化及肠道短链脂肪酸的影响

Effects of Moxibustion at "Feishu (BL13)" and "Tianshu (ST25)" on Airway Remodeling,Immune Cell Differentiation and Intestinal Short-chain Fatty Acids in Bronchial Asthma Model Rats

目的 探讨艾灸肺俞、天枢防治支气管哮喘的可能作用机制。方法 48只SD大鼠随机分为正常组12只、造模组36只。造模组大鼠采用卵清白蛋白(OVA)腹腔及皮下注射致敏联合雾化激发的方法构建支气管哮喘大鼠模型。将造模成功的32只大鼠随机分为模型组、肺俞组、天枢组和肺俞-天枢组,每组8只。正常组和模型组大鼠捆绑固定不做干预,肺俞组大鼠艾灸双侧“肺俞”30 min,天枢组大鼠艾灸双侧“天枢”30 min,肺俞-天枢组大鼠艾灸双侧“肺俞”和“天枢”各15 min。艾灸干预结束1 h后,除正常组外其余各组大鼠均给予1%OVA溶液雾化激发20 min,正常组用同体积0.9%氯化钠溶液雾化20 min。以上干预措施均每天1次,连续14天。造模后和干预期间进行行为学观察,末次干预24 h后取材,采用HE染色、Masson染色分别观察各组大鼠肺组织病理学形态变化,计算胶原纤维沉积面积百分比;ELISA法检测血清中白细胞分化抗原11b (CD11b)、白细胞分化抗原40 (CD40)、白细胞分化抗原86 (CD86)、程序性死亡配体2 (PD-L2)含量,以及肺组织中白细胞介素8 (IL-8)、白细胞介素11 (IL-11)、白细胞介素27 (IL-27)含量;Western blot法检测肺组织中基质金属蛋白酶9 (MMP-9)、基质金属蛋白酶抑制剂1 (TIMP-1)、转化生长因子β(TGF-β)蛋白表达水平;采用气相色谱-质谱联用检测器检测大鼠粪便中6种(乙酸、丙酸、异丁酸、丁酸、戊酸、正己酸)短链脂肪酸(SCFAs)含量。结果 与正常组比较,模型组大鼠逐渐出现精神萎靡或烦躁不安,毛发枯槁暗淡,活动迟缓,进食量减少,大便不成形,伴有呼吸急促、喘息症状;病理学形态结果显示肺组织结构重度异常,支气管周围可见大量炎症细胞浸润,气道平滑肌层增厚以及大量胶原纤维沉积,胶原纤维沉积面积百分比增加;大鼠血清中CD11b、CD40、CD86、PD-L2含量均升高,肺组织中IL-8、IL-11、IL-27含量和MMP-9、TGF-β、TIMP-1蛋白表达均升高,粪便中乙酸、丙酸、异丁酸、丁酸、戊酸、正己酸含量均降低(P<0.05或P<0.01)。与模型组比较,肺俞组、天枢组和肺俞-天枢组大鼠精神状态、毛发色泽、活动度、饮水进食以及呼吸急促、喘息症状均有所改善,大便基本成形,肺组织病理学形态均得到改善,大鼠血清中CD11b、CD40、CD86、PD-L2含量,肺组织中IL-8、IL-27含量和TGF-β蛋白表达,以及胶原纤维沉积面积百分比均降低;肺俞组和肺俞-天枢组肺组织中IL-11含量及MMP-9、TIMP-1蛋白表达均降低;肺俞-天枢组粪便中6种SCFAs含量均升高(P<0.05或P<0.01)。与肺俞组比较,肺俞-天枢组肺组织中胶原纤维沉积面积百分比、TIMP-1蛋白表达降低,粪便中乙酸、丁酸含量升高(P<0.05或P<0.01)。与天枢组比较,肺俞-天枢组血清中CD40含量降低,肺组织中胶原纤维沉积面积百分比、IL-11含量及MMP-9、TGF-β、TIMP-1蛋白表达降低,粪便中乙酸、丙酸、丁酸含量升高(P<0.05或P<0.01)。结论 艾灸肺俞、天枢对支气管哮喘大鼠的气道重塑有较好的治疗作用,且肺俞与天枢两穴联用具有协同作用,其作用机制可能与调节肠道SCFAs含量、影响免疫细胞分化、减轻气道炎症有关。

Objective To explore the possible mechanism of moxibustion at"Feishu(BL13)"and"Tianshu(ST25)"in treatment of bronchial asthma.Methods A total of 48 SD rats were randomly divided into normal group(n=12)and modeling group(n=36).The bronchial asthma rat model was established by sensitization with ovalbumin(OVA)injection and aerosol provocation.Thirty-two successfully modeled rats were further randomly divided into four groups including model group,Feishu group,Tianshu group,and Feishu-Tianshu group,with 8 rats in each group.Rats in the normal group and the model group were tied and fixed without intervention,while those in the Feishu group received moxibustion at bilateral of"Feishu(BL13)"for 30 minutes;rats in the Tianshu group received moxibustion at bilateral"Tianshu(ST25)"for 30 minutes,and those in the Feishu-Tianshu group received moxibustion at both"Feishu(BL13)"and"Tianshu(ST25)"bilaterally for 15 minutes each.One hour after the intervention,1%OVA solution was aerosolized for 20 minutes in all groups except the normal group,which was given the same volume of 0.9%sodium chloride solution instead of OVA solution for aerosol stimulation.The above interventions were performed once daily for 14 days.Behavioral observations were performed after modeling and during the interventions.The samples were collected 24h after the last intervention.HE and Masson staining were used to observe pathological morphological changes of lung tissues,and the percentage of collagen fiber deposition area was counted.The levels of leukocyte differentiation antigen11b(CD11b),leukocyte differentiation antigen 40(CD40),leukocyte differentiation antigen 86(CD86),and programmed death ligand 2(PD-L2)in serum,as well as the expression of interleukin-8(IL-8),interleukin-11(IL-11),interleukin-27(IL-27)in lung tissue,were detected by enzyme-linked immunosorbent assay(ELISA).Western blot was used to detect the expression of matrix metalloproteinase-9(MMP-9),matrix metalloproteinase inhibitor 1(TIMP-1)and transforming growth factorβ(TGF-β)proteins in lung tissue.The content of six short-chain fatty acids(SCFAs)including acetic acid,propionic acid,isobutyric acid,butyric acid,valeric acid,and hexanoic acid in feces was detected by gas chromatography-mass spectrometry.Results Compared to the normal group,the rats in the model group gradually showed mental depression or restlessness,dull hair,slow activity,reduced food intake,unformed stool,accompanied by symptoms of shortness of breath and wheezing.The pathological results showed severe abnormalities in lung tissue structure in the model group,including extensive infiltration of inflammatory cells around the bronchi,thickening of the airway smooth muscle layer,and substantial deposition of collagen fibers.Significant increases were observed in the levels of serum CD11b,CD40,CD86,and PD-L2,levels of IL-8,IL-11,and IL-27 in the lung tissue,as well as protein expression levels of MMP-9,TGF-β,and TIMP-1 in lung tissue,while the fecal levels of acetic acid,propionic acid,isobutyric acid,butyric acid,valeric acid,and n-caproic acid significantly decreased(P<0.05 or P<0.01).Compared to those in the model group,the spirit,hair,activity,drinking and eating condition,shortness of breath,and wheezing symptoms of rats in the Feishu group,Tianshu group,and Feishu-Tianshu group were improved;the stool was basically formed,and the pathological morphology of lung tissue were improved;the levels of serum CD11b,CD40,CD86 and PD-L2,the levels of IL-8 and IL-27 in the lung tissue,the percentage of collagen fiber deposition area,and the TGF-βprotein expression notably decreased;content of IL-11 and MMP-9 in the lung tissue and protein expression of T1MP-1 in Feishu group and Feishu-tianshu group significantly decreased;content of six SCFAs in the Feishu-Tianshu group increased(P<0.05 or P<0.01).Compared to those in the Feishu group,the percentage of collagen fiber deposition area and TIMP-1 protein expression in lung tissue in the Feishu-Tianshu group significantly decreased,while the fecal levels of acetic acid and butyric acid notably increased(P<0.05 or P<0.01).Compared to those in the Tianshu group,the serum level of CD40 in the Feishu-Tianshu group was significantly reduced,and the percentage of collagen fiber deposition area,the content of IL-11,and the protein expressions of MMP-9,TGF-βand TIMP-1 in the lung tissue notably decreased,while the fecal levels of acetic acid,propionic acid,and butyric acid significantly increased(P<0.05 or P<0.01).Conclusion Moxibustion at"Feishu(BL13)"and"Tianshu(ST25)"exhibits a favorable therapeutic effect on airway remodeling in bronchial asthma rats,and the combined application of"Feishu(BL13)"and"Tianshu(ST25)"acupoints demonstrates a synergistic effect.The mechanism may be related to the regulation of intestinal SCFAs content,influencing the differentiation of immune cells,and reducing airway inflammation.