Exploring the mechanism of moxibustion in myocardial protection of rats with long-term fatigue exercise based on the classical pyroptosis pathway

Abstract

Objective

To explore the possible mechanism of moxibustion in myocardial protection of rats undergoing long-term fatigue exercise based on observing the classical pyroptosis pathway mediated by nuclear factor kappa-B (NF-κB)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3)/cysteinyl aspartate specific proteinase 1 (Caspase-1).

Methods

A total of 50 specific-pathogen-free male Sprague-Dawley rats were bought. Ten unqualified rats were excluded, and the remaining 40 rats were divided into a normal group, a normal + Shenque (CV8) group, a model group, a model + non-meridian non-point group, and a model + Shenque (CV8) group according to the random number table method, with 8 rats in each group. Except for rats in the normal group and the normal + Shenque (CV8) group, rats in the other three groups were trained with an incline running table exercise protocol to create a long-term fatigue exercise model, 1 h/time, once a day for 5 d with 2 d off, for a total of 8 weeks. Rats in the normal group received no modeling or intervention. Rats in the normal + Shenque (CV8) group were not modeled but received mild moxibustion at Shenque (CV8); those in the model group were modeled only without intervention; those in the model + non-meridian non-point group received moxibustion at non-meridian and non-point spots after the modeling; those in the model + Shenque (CV8) group received moxibustion at Shenque (CV8) after modeling. The above moxibustion interventions were performed for 15 min/time once daily, for 5 d with 2 d off per week and a total of 8 weeks. Blood was collected from the femoral artery 4 h after the last exercise, and the serum interleukin (IL)-1β and IL-18 levels were measured. The NF-κB, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1, and gasdermin D (GSDMD) expression levels were detected by Western blotting. Myocardial morphology and pyroptosis were observed by hematoxylin-eosin (HE) staining and electron microscopy.

Results

The HE staining results showed that the myocardial cells in the model group and the model + non-meridian non-point group were disorganized with blurred transverse lines, widened interstitial spaces, interstitial edema, and inflammatory cell infiltration. The structure of myocardial cells in the model + Shenque (CV8) group was clearly visible, with slightly widened interstitial spaces and occasional infiltration of inflammatory cells in the interstitium. Compared with the normal group, the serum IL-1β and IL-18 levels were increased, and myocardial NF-κB, NLRP3, ASC, Caspase-1, and GSDMD expression levels were elevated in the model group and the model + non-meridian non-point group (P<0.01). Compared with the model group, the above indicators did not change significantly in the model + non-meridian non-point group, while all the above indicators were decreased in the model + Shenque (CV8) group (P<0.01). Compared with the model + non-meridian non-point group, all the above biochemical indicators were decreased in the model + Shenque (CV8) group (P<0.01). Transmission electron microscopy showed that the mitochondria number was increased in the model group and the model + non-meridian non-point group, some of the mitochondrial lumen was irregularly enlarged, the cell membrane structure was unclear, and chromatin was aggregated. The mitochondria number was increased, the swelling was reduced, and the nuclear membrane structure was more intact in the model + Shenque (CV8) group.

Conclusion

Moxibustion at Shenque (CV8) regulates the NF-κB/NLRP3/Caspase-1 pathway and reduces the pyroptosis in the myocardium of rats with long-term fatigue exercise, thus reducing the myocardial injury caused by long-term fatigue exercise.

摘要

目的

通过观察核因子&kappa;B(NF-&kappa;B)/核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)/半胱氨酸天冬氨酸蛋白酶1(Caspase-1)介导的经典焦亡通路探讨艾灸对长期疲劳运动大鼠心肌保护作用的可能机制。

方法

共50只无特定病原体级雄性Sprague-Dawley大鼠, 剔除不合格10只, 剩余40只按照随机数字表法分为正常组、正常+神阙组、模型组、模型+ 非经非穴组和模型+神阙组, 每组8只。除正常组和正常+神阙组外, 其余三组采用坡度跑台运动方案制造长期疲劳运动模型, 每次1 h, 每日1次, 连续训练5 d, 休息2 d, 共训练8周。正常组不造模、不干预; 正常+神阙组不造模, 但接受温和灸神阙穴; 模型组仅造模, 不干预; 模型+ 非经非穴组在造模基础上加灸非经非穴点; 模型+ 神阙组在造模基础上加灸神阙穴。以上灸法干预每次15 min, 每日1次, 干预5 d, 休息2 d, 共8周。末次运动4 h后股动脉取血, 检测血清白细胞介素(IL)-1&beta;及IL-18含量。取大鼠左心室组织, 采用免疫印迹法检测NF-&kappa;B、NLRP3、凋亡相关斑点样蛋白(ASC)、Caspase-1及焦孔素D(GSDMD)表达; 采用苏木素-伊红(HE) 染色和电镜观察心肌形态和细胞焦亡情况。

结果

HE结果显示, 模型组和模型+ 非经非穴组心肌细胞排列紊乱, 横纹模糊, 细胞间隙增宽, 间质水肿, 炎性细胞浸润。模型+神阙组心肌细胞结构清晰可见, 细胞间隙略增宽, 间质内偶见部分炎性细胞浸润。与正常组比较, 模型组、模型+ 非经非穴组的血清IL-1&beta;和IL-18含量增加, 心肌NF-&kappa;B、NLRP3、ASC、Caspase-1及GSDMD表达升高(P<0.01)。与模型组比较, 模型+ 非经非穴组上述各项指标变化不明显, 模型+神阙组上述各项指标均降低(P<0.01)。与模型+ 非经非穴组比较, 模型+神阙组上述各生化指标均下降(P<0.01)。透射电镜下观察结果提示, 模型组和模型+ 非经非穴组线粒体数目增多, 有部分线粒体腔呈不规则样变大, 细胞膜结构不清晰, 染色质聚集; 模型+神阙组线粒体数目增多, 肿胀程度减轻, 核膜结构较为完整。

结论

艾灸神阙穴可调节长期疲劳运动大鼠心肌NF-&kappa;B/NLRP3/Caspase-1通路, 减少细胞焦亡的发生, 进而减轻长期疲劳运动造成的心肌损伤。