| Objective:Discuss the effect of mTOR pathway on synthesis of cytoskeleton protein and actin in DN and whether Th17/Treg to regulate podocyte-associated protein via mTOR signaling pathway by the research of blocking mTOR signaling pathway on podocyte Ezrin,α-SMA protein and peripheral TH17/Treg cells in rats of DN.Then provide new targets for the treatment of DN.Method:Model and packet:there were 40 SD male rats and adaptive feeded one week,then divided them into 2 groups randomly:normal group(A) 10, odel 30;Model rats were injected with streptozotocin(streptozotocin,STZ)by intraperitoneal injection,dose of 55 mg / Kg,measured blood glucose after 3 days.It was a successful model when random blood glucose level≥16.7mmol/L and be stable 5 days.If no compliance,additionally injected STZ 50 mg / kg,then measured blood glucose after 3days.the successful model groups were randomly divided into DN group(group B)、DN+tacrolimus intervention group(tacrolimus FK506)(group C)、DN+ku0063794intervention group(group D),each group has 10 mices;The mices of group C and D were gavage administration by FK506 1mg/kg/d, ku0063794 1mg/kg/d respectively,Group A and B were fed normal saline solution.Detected weight, blood glucose,creatinine clearance(Ccr), 24-hour urinary protein of rats of group A,B,C and D in0,4 and 8 weeks.Detected IL-17,TGF-β1 expression of rats by ELISA;Detected the accounting of CD4+CD25hihg Treg in total CD4+T by flow cytometry.Half rats of each group were sacrificed when 4,8 weeks,and then eviscerated the kidney tissue.Detected Ezrin-mRNA,α-SMA by RT-PCR and mTOR, Raptor, Rictor, Ezrin, α-SMA protein by WB.Analyzed Analyze whether it was e statistically significant in the differences between the group and the index group by statistical methods.Results:(1)Biochemical index(1) 24 h urinary protein, comparison between the groups:At 0 week,groupB, C and D were significantly higher than group A(P <0.01),there were not significant difference between group B, C and D; At 4 weeks, groupB,C and D were obviously higher than group A(P<0.01),group C and D were lower than group B(P<0.05),there were not significant difference between group C and D; At 8weeks,group B was significantly higher than group A(P<0.001), group C and D were significant lower than group B(P<0.05),there were not significant difference between group A, C and D.Comparison within the group:8 weeks of group B was significantly higher than 0 week of group B(P<0.05);8 weeks of group C and D were significantly lower than 0 week of group C and D(P<0.05).(2) Ccr,comparison between the groups:At 4 weeks,group B was lower than group A(P<0.05),group D was higher than group B(P<0.05), group C was not significant difference with group B and D; At8 weeks,group B was significantly lower than group A(P<0.05),group C and D were significantly higher than group B(P<0.05),there were not significant difference between group C and D.There was no significant difference in the group.(2)Western blotting index(1)mTOR protein, comparison between the groups:At 4 weeks, groupB, C and D were higher than group A(P<0.05), group C was lower than group B(P <0.05), group D was not significant difference with group B and C;At 8 weeks, group B was obviously higher than group A(P<0.05),group C and D were obviously lower than group B(P<0.001),there were no significant difference between group A,C and D.There was no significant difference in the group.(2)Raptor protein, comparison between the groups:At 4 and 8 weeks,group B, C and D were higher than group A(P<0.05), group C and D were obviously lower than group B(P<0.05),there were not significant difference between group C and D.Comparison within the group:8 weeks of group C was significantly lower than 4 week of group C(P<0.05).(3)Rictor protein, comparison between the groups:At 4 weeks,group B, C and D were higher than group A(P<0.05),group C and D were obviously lower than group B(P<0.05),there were not significant difference between group C and D;At 8weeks, group B was obviously higher than group A(P<0.001),group C and D were obviously lower than group B(P<0.001),there were not significant difference between group A,C and D.There was no significant difference in the group.(4) Ezrin protein,Comparison within the group:At 4 weeks,group B, C and D were obviously lower than group A(P<0.05),groupC and D were higher than group B(P <0.05),there were not significant difference between C and D; At 8 weeks,group B was obviously lower than group A(P<0.05),group C and D were lower than group B(P<0.05),therewere not significant difference between group A,C and D.Comparison within the group:8 weeks of group B was lower than 4 week of group B(P<0.05).(5) α-SMA protein,Comparison within the groups:group B, C and D were higher than group A(P<0.05),group C and D were lower than group B(P<0.05),there were not significant difference between group C and D.At 8 weeks,group B, C and D were higher than group A(P<0.05),group C and D were obviously lower than group B(P<0.05),group D was lower than group C(P<0.05).Comparison within the group:8weeks of group B was higher than 4 weeks of group B(P<0.05);8 weeks of group C and D were lower than 4 weeks of group C and D(P<0.05)。(3)RT-PCR(1)Ezrin-mRNA, Comparison within the groups: At 4,8 weeks,group B, C and D were lower than group A(P <0.05), group C and D were higher than group B P <0.05), There were not significant difference between group C and D.There was no significant difference in the group.(2)α-SMA-mRNA, Comparison within the groups: At 4 weeks, group B, C and D were higher than group A(P <0.05),groupC and D were significantly lower than group B(P<0.001),There were not significant difference between group C and D;At 8 weeks, group B and C were higher than group A(P <0.05), group C and D were significantly lower than group B(P <0.001), group D was not significant difference with group A and C.(4)Flow Indexes:The percentage of CD4+CD25highTreg, Comparison within the groups:At 4, 8 weeks, group B was significantly lower than group A(P <0.05),groupC and D were higher than group B(P<0.05),there were not significant difference between groupA,C and D.Comparison within the group:8 and 4 weeks of group B were significantly lower than 0 week of group B(P <0.001); 4 weeks of group D was higher than 8 weeks of group D(P < 0.05).(5)ELISA indicators(1)TGF-β1, Comparison within the groups: At 4 weeks,group B and D were higher than group A(P <0.05), group D was lower than group B(P <0.05), group C was not significant difference with group A and B, and D; At 8weeks, group B was significantly higher than group A(P <0.05), group C and D were not significant difference with group A and B, group D was higher than group C(P <0.001).Comparison within the group : 4 weeks and 8 weeks of group B, C and D were higher than 0 week of group B, C and D(P <0.05), 8 weeks of D group was higher than 4 weeks of D group(P <0.05).(2)IL-17, Comparison within the groups:At4 weeks, group B and C were higher than group A(P <0.05), group C and D were lower than group B(P <0.05), group D was not significant difference with group A and C; At 8 weeks, group B and C were higher than group A(P <0.05), there were not significant difference between group C and B, group D was not significant difference with group A and B, and C. Comparison within the group: 8 weeks of group A was higher than 0 week of group A(P <0.05);4 weeks and 8 weeks of group B, C and D were higher than 0 week of group B, C and D(P <0.05).(6)Correlation analysis(1)In group B,Rictor was positively correlated withα-SMA-mRNA,but negatively correlated with Ezrin and Ezrin-mRNA at 4 weeks,Rictor was positively correlated with α-SMA,TGF-β1 and IL-17,but negatively correlated with Ezrin and CD4+CD25hihg Treg at 8 weeks;Raptor had not correlation with Ezrin, Ezrin-mRNA, α-SMA, α-SMA-mRNA, CD4 + CD25 high Treg, TGF-β1,IL-17.(2)In group B,CD4+ CD25 hihg Treg was negatively correlated with TGF-β1protein at 4 and 8 weeks.(3)In group B,Ezrin-mRNA was positively correlated with CD4+CD25hihgTreg at 4 and 8 weeks.Ezri and Ezrin-mRNA was negatively correlated with TGF-β1 、 IL-17 at 8 weeks.(4)In group B,α-SMA-mRNA was positively correlated with IL-17,but negatively correlated with CD4+ CD25 hihg Treg at4 weeks.Conclusion:1 It can increase glomerular filtration rate,reduce proteinuria, improve mental state in rats of DN by blocking mTOR pathway.2 mTORC2 signaling pathway participate in the expression of podocytes Ezrin,α-SMA in DN.3 Treg may regulate Ezrin synthesis, α-SMA protein by mTORC2 signaling pathway.4 FK506 blocks mTORC1 and mTORC2 signaling pathways,Simultaneously. |
