吴茱萸碱调节Nod样受体蛋白3信号通路对非酒精性脂肪性肝病大鼠肝组织损伤的影响

Influence of evodiamine on liver injury in rats with non-alcoholic fatty liver disease through the regulation of NLRP3 signal pathway

目的探讨吴茱萸碱调节Nod样受体蛋白3(NLRP3)信号通路影响非酒精性脂肪性肝病(NAFLD)大鼠肝组织损伤情况。方法2022年5—11月选取大鼠喂食高脂饲料8周后,通过随机数字表法将大鼠分为模型组、吴茱萸碱低剂量组(吴茱萸碱4 mg/kg)、吴茱萸碱中剂量组(吴茱萸碱8 mg/kg)、吴茱萸碱高剂量组(吴茱萸碱16 mg/kg)、阳性药组(多烯磷脂酰胆碱200 mg/kg)和NLRP3组(吴茱萸碱16 mg/kg+1 mg/kg NLRP3信号通路激活剂尼日利亚菌素),每组各10只,另有10只大鼠喂食普通饲料作为对照组,所有大鼠均给予相对应药物干预,给药4周;生化分析仪检测血清谷丙转氨酶(GPT)、谷草转氨酶(GOT)、胆固醇、三酰甘油(TG);HE染色观察肝组织病理;油红O染色观察肝组织脂肪沉积;酶联免疫吸附测定(ELISA)检测大鼠血清肿瘤坏死因子α(TNF-α)、白细胞介素(IL)-18、IL-1β水平;实时荧光定量逆转录聚合酶链反应(qRT-PCR)和蛋白质印迹法检测大鼠肝脏组织NLRP3、凋亡相关斑点样蛋白(ASC)、胱天蛋白酶-1(caspase-1)表达水平。结果与对照组相比,模型组大鼠肝组织肝小叶结构紊乱,肝细胞增大,可见明显颗粒状脂滴以及脂质积累,并伴有炎症细胞浸润,肝组织脂肪变性明显,大鼠体质量[(582.65±16.25)g比(476.29±10.62)g]、肝指数[(3.79±0.25)%比(2.48±0.18)%]、血清GPT[(79.62±3.59)U/L比(36.22±2.15)U/L]、GOT[(185.25±5.18)U/L比(71.69±3.56)U/L]、胆固醇[(2.93±0.19)mmol/L比(1.72±0.12)mmol/L]、TG[(1.19±0.11)mmol/L比(0.56±0.07)mmol/L]、TNF-α[(162.48±4.25)ng/L比(41.76±2.49)ng/L]、IL-18[(135.75±3.37)ng/L比(23.52±2.02)ng/L]、IL-1β[(201.88±4.67)ng/L比(92.64±2.99)ng/L]水平,肝组织NLRP3、ASC、caspase-1 mRNA表达以及NLRP3、ASC、活化胱天蛋白酶-1(cleaved caspase-1)蛋白表达均明显升高(P<0.05);与模型组相比,吴茱萸碱低、中、高剂量组和阳性药组大鼠肝组织损伤、肝脏脂肪变性、炎症细胞浸润和脂滴积累明显减轻,大鼠体质量[(551.37±15.72)g、(530.52±15.49)g、(509.48±15.18)g、(517.71±12.73)g比(582.65±16.25)g]、肝指数[(3.41±0.20)%、(3.13±0.16)%、(2.81±0.17)%、(3.02±0.213)%比(3.79±0.25)%]、血清GPT[(68.16±3.41)U/L、(55.94±3.05)U/L、(46.45±2.72)U/L、(48.71±2.34)U/L比(79.62±3.59)U/L]、GOT[(161.32±4.73)U/L、(138.64±4.50)U/L、(113.57±4.05)U/L、(121.48±4.11)U/L比(185.25±5.18)U/L]、胆固醇[(2.58±0.17)mmol/L、(2.25±0.16)mmol/L、(1.91±0.13)mmol/L、(1.99±0.15)mmol/L比(2.93±0.19)mmol/L]、TG[(1.01±0.10)mmol/L、(0.83±0.08)mmol/L、(0.62±0.07)mmol/L、(0.70±0.09)mmol/L比(1.19±0.11)mmol/L]、TNF-α[(137.15±3.69)ng/L、(113.53±3.34)ng/L、(79.37±2.92)ng/L、(85.61±3.07)ng/L比(162.48±4.25)ng/L]、IL-18[(111.34±3.05)ng/L、(72.98±2.66)ng/L、(47.61±2.438)ng/L、(51.59±2.55)ng/L比(135.75±3.37)ng/L]、IL-1β[(171.52±4.34)ng/L、(152.23±4.02)ng/L、(129.95±3.51)ng/L、(517.71±12.73)ng/L比(136.76±3.73)ng/L]水平,肝组织NLRP3、ASC、caspase-1 mRNA表达以及NLRP3、ASC、cleaved caspase-1蛋白表达均明显降低(P<0.05);NLRP3信号通路激活剂尼日利亚菌素的加入明显减弱了吴茱萸碱对NAFLD大鼠肝组织的保护作用。结论吴茱萸碱可通过抑制NLRP3信号通路明显改善NAFLD大鼠肝组织损伤、脂肪病变和炎症反应。

Objective To investigate the influence of evodiamine on the liver injury in rats with non-alcoholic fatty liver disease(NAFLD)by regulating the Nod-like receptor protein 3(NLRP3)signal pathway.Methods From May 2022 to November 2022,rats were randomly divided into model group,low-dose evodiamine group(4 mg/kg evodiamine),middle-dose evodiamine group(8 mg/kg evodiamine),high-dose evodiamine group(16 mg/kg evodiamine),positive group(200 mg/kg polyene phosphatidylcholine)and NLRP3 group(16 mg/kg evodiamine+1 mg/kg NLRP3 signal pathway activator Nigericin)by the randomized numerical table method after fed with high-fat diet for 8 weeks,another 10 rats were fed with common diet as the control group,all rats were given corresponding drug intervention for 4 weeks;biochemical analyzer was used to detect serum glutamic-pyruvic transaminase(GPT),glutamic-oxaloacetic transaminase(GOT),cholesterol and triglyceride(TG);HE staining was used to observe liver histopathology;oil red O staining was used to observe fatty deposition in liver tissue;ELISA was used to detect the levels of serum TNF-α,IL-18 and IL-1βin rats;Real-time quantitative PCR(qRT-PCR)and Western blotting were used to detect the expression levels of NLRP3,ASC and caspase-1 in rat liver tissues.Results Compared with the control group,the liver tissue of rats in the model group had disturbed hepatic lobule structure,enlarged hepatocytes,visible granular lipid droplets and lipid accumulation,accompanied by inflammatory cell infiltration,obvious steatosis of liver tissue,and the body mass[(582.65±16.25)g vs.(476.29±10.62)g],liver index[(3.79±0.25)%vs.(2.48±0.18)%],serum GPT[(79.62±3.59)U/L vs.(36.22±2.15)U/L],GOT[(185.25±5.18)U/L vs.(71.69±3.56)U/L],cholesterol[(2.93±0.19)mmol/L vs.(1.72±0.12)mmol/L],TG[(1.19±0.11)mmol/L vs.(0.56±0.07)mmol/L],TNF-α[(162.48±4.25)ng/L vs.(41.76±2.49)ng/L],IL-18[(135.75±3.37)ng/L vs.(23.52±2.02)ng/L],IL-1βlevels[(201.88±4.67)ng/L vs.(92.64±2.99)ng/L],the expression of NLRP3,ASC,caspase-1 mRNA,and the expression of NLRP3,ASC,cleaved caspase-1 protein in liver tissue of rats were obviously increased(P<0.05);compared with the model group,the liver tissue damage,liver steatosis,inflammatory cell infiltration and lipid droplet accumulation of rats in the low,middle and high-dose evodiamine groups and the positive group were obviously reduced,the body mass[(551.37±15.72)g,(530.52±15.49)g,(509.48±15.18)g,(517.71±12.73)g vs.(582.65±16.25)g],liver index[(3.41±0.20)%,(3.13±0.16)%,(2.81±0.17)%,(3.02±0.213)%vs.(3.79±0.25)%],serum GPT[(68.16±3.41)U/L,(55.94±3.05)U/L,(46.45±2.72)U/L,(48.71±2.34)U/L vs.(79.62±3.59)U/L],GOT[(161.32±4.73)U/L,(138.64±4.50)U/L,(113.57±4.05)U/L,(121.48±4.11)U/L vs.(185.25±5.18)U/L],cholesterol[(2.58±0.17)mmol/L,(2.25±0.16)mmol/L,(1.91±0.13)mmol/L,(1.99±0.15)mmol/L vs.(2.93±0.19)mmol/L],TG[(1.01±0.10)mmol/L,(0.83±0.08)mmol/L,(0.62±0.07)mmol/L,(0.70±0.09)mmol/L vs.(1.19±0.11)mmol/L)],TNF-α[(137.15±3.69)ng/L,(113.53±3.34)ng/L,(79.37±2.92)ng/L,(85.61±3.07)ng/L vs.(162.48±4.25)ng/L],IL-18[(111.34±3.05)ng/L,(72.98±2.66)ng/L,(47.61±2.438)ng/L,(51.59±2.55)ng/L vs.(135.75±3.37)ng/L],IL-1βlevels[(171.52±4.34)ng/L,(152.23±4.02)ng/L,(129.95±3.51)ng/L,(517.71±12.73)ng/L vs.(136.76±3.73)ng/L],the expression of NLRP3,ASC,caspase-1 mRNA,and the expression of NLRP3,ASC,Cleared caspase-1 protein in liver tissue of rats were obviously decreased(P<0.05);the addition of Nigericin,an activator of NLRP3 signaling pathway,ob-viously weakened the protective effect of evodiamine on liver tissue of NAFLD rats.Conclusion Evodiamine can obviously improve the liver tissue injury,fatty lesion and inflammatory reaction in NAFLD rats by inhibiting the NLRP3 signal pathway.