基于TXNIP/NLRP3信号通路研究根皮素对糖尿病肾病小鼠肾脏自噬和纤维化的影响

Phloretin Regulates Renal Autophagy and Fibrosis in Mouse Model of Diabetic Nephropathy via TXNIP/NLRP3 Pathway

目的:基于硫氧还蛋白相互作用蛋白/核苷酸结合寡聚化结构域样受体蛋白3(TXNIP/NLRP3)信号通路研究根皮素对糖尿病肾病(DN)小鼠肾脏自噬和纤维化的影响及分子机制。方法:采用腹腔注射链脲佐菌素30 mg/kg建立DN小鼠模型。随机将小鼠分为正常对照组、模型对照组、吡格列酮10 mg/kg组、根皮素200、400 mg/kg组,每组10只。小鼠连续灌胃根皮素10 w后,测定各组小鼠肾脏肥大指数,采用HE染色、PAS染色和Masson染色观察小鼠肾脏病理形态学变化和肾纤维化改变程度,检测小鼠血糖、体质量、饮水量、尿量、尿β2微球蛋白(β2-MG)含量,取血清测定尿素氮(BUN)、肌酐(Scr)含量,酶联免疫吸附法(ELISA)测定血清中白细胞介素-1(IL-1)、IL-18、转化生长因子-β1(TGF-β1)含量;采用免疫组化法测定肾组织盘状结构域受体1(DDR1)、TXNIP、NLRP3、IL-1β、自噬效应蛋白Beclin-1、微管相关蛋白1轻链3-Ⅱ(LC3-Ⅱ)蛋白表达,并用Western blot法检测肾脏TGF-β1、α-平滑肌肌动蛋白(α-SMA)、Beclin-1、LC3-Ⅱ/LC3-Ⅰ蛋白表达。结果:与正常对照组比较,模型对照组小鼠血糖、血清BUN、Scr、IL-1、IL-18、TGF-β1、尿β2-MG含量显著升高,肾脏肥大指数显著增加(P<0.01);肾脏DDR1、TXNIP、NLRP3、IL-1β、TGF-β1、α-SMA蛋白表达显著上调(P<0.01),Beclin-1显著上调、LC3-Ⅱ/LC3-Ⅰ比值显著降低(P<0.01);与模型对照组比较,根皮素200、400 mg/kg组和吡格列酮10 mg/kg组小鼠血糖、血清BUN、Scr、IL-1、IL-18、TGF-β1、尿β2-MG含量显著降低,肾脏肥大指数显著降低(P<0.01);肾脏DDR1、TXNIP、NLRP3、IL-1β、TGF-β1、α-SMA蛋白表达显著下调(P<0.01),Beclin-1蛋白表达上调、LC3-Ⅱ/LC3-Ⅰ比值显著升高(P<0.01),肾脏病理形态和纤维化明显改善。结论:根皮素能提高肾脏自噬水平并抑制肾纤维化,其机制可能与调控TXNIP-NLRP3信号通路减轻炎症反应有关。

Objective:To study the effects of phloretin on renal autophagy and fibrosis in the mouse model of diabetic nephropathy(DN)and decipher the molecular mechanism via the thioredoxin-interacting protein(TXNIP)/nucleotide-binding oligomerization domain,leucine-rich repeat and pyrin domain-containing protein 3(NLRP3)pathway.Methods:The mouse model of DN was established by intraperitoneal injection of 30 mg/kg streptozotocin.Mice were randomized into normal control,model,pioglitazone(10 mg/kg),phloretin(200 mg/kg and 400 mg/kg)groups,with 10 mice in each group.After 10 continuous weeks of intervention with phloretin by gavage,the renal hypertrophy index was measured.HE staining,PAS staining,and Masson staining were employed to observed the pathomorphological changes of mice kidneys and the degree of renal fibrosis.The blood glucose,body weight,water consumption,urine volume,and urineβ2-microglobulin(β2-MG)of mice were measured.The serum samples were collected for the measurement of blood urea nitrogen(BUN)and serum creatinine(Scr)levels.Enzymelinked immunosorbent assay was employed to measure the serum levels of interleukin(IL)-1,IL-18,and transforming growth factor(TGF)-β1.The immunohistochemical method was used to examine the expression of discoidin domain receptor 1(DDR1),TXNIP,NLRP3,IL-1β,beclin-1,and microtubule-associated protein light chain II(LC3-Ⅱ)in the renal tissue.Western blotting was employed to determine the protein levels of TGF-β1,α-smooth muscle actin(SMA),beclin-1,and LC3-Ⅱ/LC3-Ⅰin the renal tissue.Results:Compared with the normal group,the modeling elevated the blood glucose,BUN,Scr,IL-1,IL-18 TGF-β1,andβ2-MG levels,and increased the renal hypertrophy index(P<0.01).Furthermore,the modeling of DN up-regulated the protein levels of DDR1,TXNIP,NLRP3,IL-1β,TGF-β1,andα-SMA(P<0.01),promoted the expression of beclin-1,and decreased the LC3-Ⅱ/LC3-Ⅰratio(P<0.01)in the renal tissue kidney.Compared with the model group,the drug interventions lowered blood glucose,BUN,Scr,IL-1,IL-18,TGF-β1,andβ2-MG levels and decreased the renal hypertrophy index(P<0.01).Moreover,the drug interventions down-regulated the protein levels of DDR1,TXNIP,NLRP3,IL-1β,TGF-β1,andα-SMA(P<0.01),promoted the expression of beclin-1,and increased the LC3-Ⅱ/LC3-Ⅰratio(P<0.01).In addition,the pathological changes and fibrosis of the kidney were mitigated in the drug intervention groups.Conclusion:Phloretin can improve the autophagy level and inhibit fibrosis in the renal tissue by regulating the TXNIP-NLRP3 pathway to reducing inflammation.