Objective: To observe the effect and mechanism of electroacupuncture (EA) on non-canonical pathway of hepatocellular pyroptosis in nonalcoholic fatty liver disease (NAFLD).
Methods: Sixty male SD rats were randomly divided into a normal diet group (n=15) and a high fat modeling group (n=45). The rats in the high fat modeling group were fed with customized high fat diet for 8 weeks to establish NAFLD model. Thirty successfully modeled rats were selected and randomly divided into a model group (n=10), an EA group (n=10) and a non-acupoint with shallow needling group (n=10), and 10 rats were randomly selected from the normal diet group as the control group additionally. In the EA group, EA was applied at bilateral "Fenglong" (ST 40) and "Ganshu" (BL 18), with disperse-dense wave, in frequency of 4 Hz/20 Hz and in intensity of 3 mA. In the non-acupoint with shallow needling group, shallow needling was delivered at points 5 mm from bilateral "Fenglong" (ST 40) and "Ganshu" (BL 18), the EA stimulation parameters were same as the EA group. The intervention was given once a day, 20 min a time, 5 days a week for 4 weeks in the two groups. After intervention, the liver morphology was observed by oil red "O" staining, the serum levels of lipopolysaccharide (LPS), interleukin (IL)-1β, IL-18 and tumor necrosis factor-α (TNF-α) were detected by ELISA, the protein expression of gasdermin D (GSDMD), GSDMD-N, cysteine aspartic acid specific protease-11 (Caspase-11), IL-1β, IL-18 and TNF-α in liver tissue were detected by Western blot, the mRNA expression of GSDMD, Caspase-11, IL-1β, IL-18 and TNF-α in liver tissue was detected by real-time PCR in rats of each group.
Results: In the model group, vacuoles in different size were found in the hepatocellular cytoplasm, and the fat droplets were in schistose accumulation. Compared with the model group, the hepatocellular fat droplets and the degree of hepatic steatosis were reduced in the EA group and the non-acupoint with shallow needling group. Compared with the control group, the serum levels of LPS, IL-1β, IL-18 and TNF-α were increased (P<0.01), the protein and mRNA expression of GSDMD, Caspase-11, IL-1β, IL-18, TNF-α as well as the protein expression of GSDMD-N in the liver tissue were increased (P<0.01) in the model group. Compared with the model group, the serum levels of LPS, IL-1β, IL-18 and TNF-α were decreased (P<0.01), the protein and mRNA expression of GSDMD, IL-1β, IL-18 and TNF-α in the liver tissue were decreased (P<0.01), the protein expression of GSDMD-N and the mRNA expression of Caspase-11 in the liver tissue were decreased (P<0.01) in the EA group and the non-acupoint with shallow needling group. Compared with the model group, the protein expression of Caspase-11 in the liver tissue was decreased (P<0.01) in the EA group. Compared with the non-acupoint with shallow needling group, the serum levels of LPS, IL-1β, IL-18 and TNF-α were decreased (P<0.01), the protein and mRNA expression of GSDMD, Caspase-11, IL-1β and IL-18 in the liver tissue were decreased (P<0.01), the protein expression of GSDMD-N and the mRNA expression of TNF-α in the liver tissue were decreased (P<0.01) in the EA group.
Conclusion: EA can inhibit hepatocellular pyroptosis in NAFLD rats, and its mechanism may be related to reducing the serum level of LPS, and down-regulating the expression of the non-canonical pathway related factors i.e. GSDMD, GSDMD-N, Caspase-11, IL-1β, IL-18 and TNF-α.
目的：观察电针对非酒精性脂肪性肝病（NAFLD）肝细胞焦亡非经典途径的影响及其作用机制。方法：将60只雄性SD大鼠随机分为普通饮食组（n=15）和高脂造模组（n=45），高脂造模组以定制高脂饲料喂养8周建立NAFLD模型。从造模成功的大鼠中选取30只随机分为模型组（n=10）、电针组（n=10）和非穴浅刺组（n=10），另从普通饮食组中随机选取10只大鼠作为对照组。电针组大鼠于双侧“丰隆”“肝俞”穴行电针干预，予疏密波，频率4 Hz/20 Hz，电流强度3 mA；非穴浅刺组大鼠于双侧“丰隆”“肝俞”穴旁5 mm处浅刺，电针刺激参数同电针组。两组干预均每日1次，每次20 min，每周5 d，连续干预4周。干预后，采用油红“O”染色法观察各组大鼠肝脏形态；ELISA法检测各组大鼠血清脂多糖（LPS）、白细胞介素（IL）-1β、IL-18以及肿瘤坏死因子-α（TNF-α）含量；Western blot法检测各组大鼠肝脏组织消皮素D（GSDMD）、GSDMD末端氨基结构域（GSDMD-N）、半胱氨酸天冬氨酸蛋白酶（Caspase）-11、IL-1β、IL-18及TNF-α蛋白表达；实时荧光定量PCR法检测各组大鼠肝脏组织GSDMD、Caspase-11、IL-1β、IL-18及TNF-α mRNA表达。结果：模型组大鼠肝细胞胞质中可见大小不等的空泡，脂滴呈片状堆积；与模型组比较，电针组及非穴浅刺组大鼠肝细胞脂滴减少，肝脏脂肪变性程度降低。与对照组比较，模型组大鼠血清LPS、IL-1β、IL-18、TNF-α含量升高（P<0.01）；肝脏组织GSDMD、Caspase-11、IL-1β、IL-18、TNF-α蛋白和mRNA表达升高（P<0.01），GSDMD-N蛋白表达升高（P<0.01）。与模型组比较，电针组及非穴浅刺组大鼠血清LPS、IL-1β、IL-18、TNF-α含量降低（P<0.01），肝脏组织GSDMD、IL-1β、IL-18、TNF-α蛋白及mRNA表达降低（P<0.01），肝脏组织GSDMD-N蛋白表达及Caspase-11 mRNA表达降低（P<0.01）；电针组大鼠肝脏组织Caspase-11蛋白表达降低（P<0.01）。与非穴浅刺组比较，电针组大鼠血清LPS、IL-1β、IL-18、TNF-α含量降低（P<0.01）；肝脏组织GSDMD、Caspase-11、IL-1β、IL-18蛋白和mRNA表达降低（P<0.01），GSDMD-N蛋白及TNF-α mRNA表达降低（P<0.01）。结论：电针干预可抑制NAFLD大鼠肝细胞焦亡，其作用机制可能与降低血清LPS含量，下调细胞焦亡非经典途径相关因子GSDMD、GSDMD-N、Caspase-11、IL-1β、IL-18、TNF-α表达相关。.
Keywords: electroacupuncture; gasdermin D (GSDMD); nonalcoholic fatty liver disease; pyroptosis.