槲皮素对骨髓间充质干细胞移植联合肾移植所诱导的免疫耐受的影响

Effects of quercetin on immune tolerance induced by bone marrow mesenchymal stem cells in kidney transplantation

目的探讨槲皮素对骨髓间充质干细胞(BMSCs)诱导肾移植免疫耐受的影响。方法在对肾移植后的大鼠给予BMSCs治疗的基础上,同时给予不同剂量的槲皮素,观察其诱导免疫耐受的效果。建立大鼠肾移植模型,随机分为4组:对照组、BMSC组、MSC+高/低剂量槲皮素干预组。Western blot检测各组肾组织中Janus激活激酶(JAK)、信号转导子和转录激活子3(STAT3)、p-STAT3的蛋白表达水平,ELISA检测血清中转化生长因子β1(TGF-β1)水平。收集BMSC组肾小管上皮细胞,干扰及过表达STAT3的同时予以槲皮素干预,使用Western blot检测JAK、STAT3、p-STAT3及TGF-β1表达。采用单因素方差分析比较各组间数据差异,组间数据两两比较采用SNK-q检验。结果大鼠经肾移植后,对照组血清中TGF-β1蛋白表达(19162±223)pg/ml高于其他组,BMSC组(12 106±163)pg/ml、MSC+高槲皮素干预组(9 113±110)pg/ml、MSC+低槲皮素干预组(9 024±133)pg/ml。3组分别与对照组比较P均<0.01。对照组肾组织中JAK、p-STAT3的蛋白相对表达量(1.5±0.2,10.7±0.5)均高于其他各组[MSC组(0.9±0.3,8.5±0.2;P均<0.01)、MSC+高槲皮素干预组(0.7±0.2,6.1±0.3;P均<0.01)、MSC+低槲皮素干预组(0.7±0.3,5.8±0.1;P均<0.01)]。干扰STAT3后肾小管上皮细胞中p-STAT3和TGF-β1蛋白相对表达量(0.7±0.1,0.3±0.1)均少于其他各组[对照组(8.2±0.4,7.1±0.5;P均<0.01)、槲皮素干预组(5.6±0.3,4.8±0.4;P均<0.01)、空白对照组(5.7±0.3,4.5±0.4;P均<0.01)]。过表达STAT3后中p-STAT3和TGF-β1蛋白相对表达量(10.5±0.6,8.4±0.5)高于槲皮素干预组[(5.7±0.3,5.4±0.3);P均<0.01]和空白对照组[(6.1±0.1,5.6±0.4);P均<0.01]。结论大鼠肾移植时在给予BMSCs注射的同时给予槲皮素干预,可有效地抑制免疫排斥反应,改善肾功能;JAK/STAT3/TGF-β1可能参与了槲皮素诱导的免疫调节。

Objective The study was to investigate the effects of different concentrations of quercetin on bone marrow mesenchymal stem cells(BMSCs)-induced immune tolerance in rats after kidney transplantation.Methods he rats were randomly divided into four groups after kidney transplantation:control group,BMSCs group,MSC+low-dosage quercetin group and MSC+high-dosage quercetin group.Janus-activated kinase(JAK),Signal transducer and activator of transcription 3(STAT3),p-STAT3 protein expression in kidney tissue were detected by Western blot.Serum transforming growth factor-P1(TGF-β1)was detected by ELISA assay.We collected the proximal tubule epithelial cells from the MSC group,and cultured them with or without stimulation of quercetin.Expressions of JAK,STAT3,p-STAT3,and TGF-β1 were detected by Western blot after STAT3 was inhibited and overexpressed in these cells,respectively.Results After kidney transplantation,TGF-pl in serum in the control group(19 162±223)pg/ml was higher than any of other groups(BMSC group,12 106±163 pg/ml;MSC+high dosage of quercetin groups,9 113±110 pg/ml;MSC+low dosage of quercetin groups,9 024±133 pg/ml).When compared to the control group,the q values of these groups were 96.88,138.0,139.2,respectively(P<0.01).Protein expression of JAK and p-STAT3 in the kidney tissue(1.5±0.2,10.7±0.5)were significantly higher in control group than in the other groups:BMSC group(0.9±0.3,8.5±0.2;P=0.0059),MSC+high dosage of quercetin groups(0.7±0.2,6.1±0.3;P=0.0002),and MSC+low dosage of quercetin groups(0.7±0.3,5.8±0.1;P=0.0011).In group with inhibition of STAT3,relative protein expression of p-STAT3 and TGF-β1(0.7±0.1,0.3±0.1)in the proximal tubule epithelial cells were significantly reduced compared to the other groups:control group(8.2±0.4,7.1±0.5;P<0.0001,P<0.0001),quercetin group(5.6±0.3,4.8±0.4;P<0.0001,P<0.0001),and si-control group(5.7±0.3,4.5±0.4;P<0.0001,P<0.0001).In group with overexpression of STAT3,relative protein expression of p-STAT3 and TGF-β1(10.5±0.6,8.4±0.5)were significantly higher than those in the quercetin group(5.7±0.3,5.4±0.3;P<0.0001,P<0.0001)and vector plasmid group(6.1±0.1,5.6±0.4;P<0.0001,P=0.0001).Conclusion Simultaneous administration of quercetin and transplantation of bone marrow mesenchymal stem cells can effectively inhibit rejection and improve renal function in rats with kidney transplantation.Quercetin may regulate immune function through JAK/STAT3/TGF-β1 pathway.