G蛋白耦联受体55拮抗剂CID16020046在小鼠肾脏纤维化中的作用

Effects of G protein-coupled receptor 55 antagonist CID16020046 on renal fibrosis in mice

目的探讨G蛋白耦联受体55(G protein-coupled receptor 55,GPR55)拮抗剂CID16020046在小鼠肾脏纤维化中的作用,为肾脏纤维化治疗提供新的方法和思路。方法(1)在体外大鼠肾脏成纤维细胞(NRK-49F)中分别过表达GPR55和使用GPR55拮抗剂CID16020046,同时使用转化生长因子β1(TGF-β1)刺激,观察纤维化相关因子和炎性因子的表达。(2)体内构建单侧输尿管梗阻(unilateral ureteral obstruction,UUO)小鼠肾脏纤维化模型,将8周龄雄性C57BL/6J小鼠(20~25 g)按照随机数字表法随机分为3组:假手术组(Sham组,n=6)、造模组(UUO组,n=7)、造模+CID16020046药物组(UUO+CID组,n=8),UUO+CID组在造模前1 d、造模当日和术后每日均腹腔注射药物CID16020046(10 mg/kg),每日1次,Sham组和UUO组均腹腔注射对应剂量的0.9%生理盐水。UUO术后7 d处死小鼠取材,检测其肾功能指标、肝脏转氨酶、心肌标志物,Western印迹和实时荧光定量PCR检测肾脏纤维化相关因子和炎性因子的表达,免疫组化、天狼猩红染色、Masson三色染色检测肾组织的病理改变。结果(1)使用TGF-β1刺激NRK-49F细胞后,GPR55 mRNA和蛋白表达量均明显增加(均P<0.05);TGF-β1组和TGF-β1+GPR55过表达质粒组纤维化相关因子纤连蛋白、Ⅰ型胶原蛋白(CollagenⅠ)和炎性因子白细胞介素1β、肿瘤坏死因子αmRNA表达的差异均无统计学意义(均P>0.05);与TGF-β1组比较,TGF-β1+CID组纤维化相关因子α平滑肌肌动蛋白(α-SMA)、波形蛋白(Vimentin)蛋白及CollagenⅠ、α-SMA mRNA表达均较低(均P<0.05)。(2)与Sham组比较,UUO组GPR55 mRNA和蛋白表达均较高(均P<0.05)。与UUO组比较,UUO+CID组血清肌酐较低(P<0.05),两组血尿素氮、谷丙转氨酶、谷草转氨酶、乳酸脱氢酶、肌酸激酶同工酶的差异均无统计学意义(均P>0.05)。与UUO组比较,UUO+CID组肾组织纤维化相关因子纤连蛋白、CollagenⅠ、Vimentin蛋白及纤连蛋白、CollagenⅠ、Ⅲ型胶原蛋白、α-SMA mRNA表达均较低,肾小管扩张和间质胶原纤维沉积程度显著较轻(均P<0.05)。结论CID16020046可降低UUO小鼠血清肌酐,保护肾功能,同时可降低肾脏成纤维细胞和小鼠肾组织纤维化相关因子表达,减轻肾脏纤维化。

Objective To explore the effects of G protein-coupled receptor 55(GPR55)antagonist CID16020046 on renal fibrosis in mice,and provide a new method and idea for the treatment of renal fibrosis.Methods(1)GPR55 overexpression and GPR55 antagonist CID16020046 were used in renal fibroblasts(NRK-49F)of rats,respectively.Meanwhile,transforming growth factor-β1(TGF-β1)was applied in the NRK-49F cells to observe the expression of fibrosis-related factors and inflammatory factors.(2)A mouse model of renal fibrosis with unilateral ureteral obstruction(UUO)was established in vivo.Eight-week-old male C57BL/6J mice(20-25 g)were randomly divided into three groups according to the random number table method:sham group(n=6),model group(UUO group,n=7),model+CID16020046 drug(UUO+CID group,n=8).The drug CID16020046(10 mg/kg)was intraperitoneally injected 1 day before modeling,on the day of modeling and every day after surgery in UUO+CID group,and the corresponding dose of 0.9%normal saline was injected intraperitoneally in sham and UUO groups.The mice were sacrificed for sampling 7 days after UUO surgery,and their renal function indicators,liver transaminase,and cardiac markers were examined.Western blotting and quantitative real-time PCR were used to detect the expression of renal fibrosis-related factors and inflammatory factors.Immunohistochemistry staining,Sirius red staining and Masson trichrome staining were used to detect the pathological changes of renal tissues.Results(1)After NRK-49F cells were stimulated by TGF-β1,the mRNA and protein expression levels of GPR55 were significantly increased(both P<0.05).There was no statistically significant difference in the mRNA expression of fibrosis-related factors fibronectin and collagenⅠ,and inflammatory factors interleukin-1βand tumor necrosis factor-αbetween TGF-β1 group and TGF-β1+GPR55 overexpression group(all P>0.05).Compared with the TGF-β1 group,the protein expression levels of fibrosis-related factors alpha-smooth muscle actin(α-SMA)and vimentin,and the mRNA expression levels of collagenⅠandα-SMA were lower in the TGF-β1+CID group(all P<0.05).(2)Compared with sham group,the mRNA and protein expression levels of GPR55 in UUO group were higher(both P<0.05).The serum creatinine in the UUO+CID group was lower compared to the UUO group(P<0.05).There was no statistically significant difference in blood urea nitrogen,alanine aminotransferase,aspartate aminotransferase,lactate dehydrogenase and creatine kinase isoenzyme between UUO group and UUO+CID group(all P>0.05).Compared with the UUO group,the protein expression levels of renal fibrosis-related factors fibronectin,collagenⅠand vimentin,and the mRNA expression levels of fibronectin,collagen Ⅰ,collagen Ⅲ and α-SMA were lower in the UUO+CID group(all P<0.05).The degree of renal tubular dilation and interstitial collagen fiber deposition in the UUO+CID group was significantly reduced compared to the UUO group(all P<0.05).Conclusions CID16020046 can reduce serum creatinine in UUO mice,protect renal function,and simultaneously decrease the expression of fibrosis-related factors in renal fibroblasts and mouse kidney tissues,thereby alleviating renal fibrosis.