Alisma Orientalis Inhibit C3 Mediating Epithelial-to-mesenchymal Transition In Renal Tubular Epithelial Cells

Objective:To observe the influence of Alisma orientalis inhibiting on C3 mediating renal tubular epithelial cells mesenchymal transition .Methods:(1)Cell experinmentsHuman proximal tubular epithelial cells(HK-2)were divided six groups according to different factors:Control group, C3a group,C3a+Alisol B 0.1umol/l group,C3a+Alisol B 1umol/l group,C3a+Alisol B 10umol/l group,C3a+Alisol B 100umol/l group.The expression of C3 mRNA were analysed by RT-PCR.Human proximal tubular epithelial cells(HK-2)were divided five groups according to different factors:Control group, C3a group,C3a+Alisol B 10umol/l group ,Alisol B 10umol/l group,TGF-βgroup。The expression of C3,a-SMA,E-cadherinmRNA and protein were analysed by RT-PCR and Western Blot.. The expression of C3,α-SMA and E-cadherin were detected by indirect immunofluorescence.(2)Animal experinments24 Sprague-Dawley male rats were randomly divided into sham-operated rats group(SOR group),unilateral ureteral obstruction model group(UUO group),Alisma orientalis-treated group.From the third day before operation,Alisol-treated group were administrated daily by intregastric administration (ia) at dose of Alisma orientalis 9k.kg.d. UUO and SOR groups were administrated a certain volumn of saline by ia. Rats were sacrificed in day 14th after operation .The kidneys removed were fixed in 10% phosphate buffered formalin. Kidney sections from paraffin embedded tissues were prepared at 4um thickness using a routine procedure.HE and Masson stain were employed to observe renal histological changes. immunohistochemical methods were used to analyze pathological changes and C3,a-SMA,E-cadherin in renal tissue. Results:(1)Compared with control group,the expression of C3mRNA in C3a group increased,while Alisol B inhibit the expression of C3Mrna with a dose-dependent manner.When the Alisol B was 10μM,the expression of C3mRNA decreased significantly.(2) RT-PCR and westernblot showed that the expression of C3,a-SMAmRNA and protein increased in HK2 cells(p<0.01), the expression of E-cadherinmRNA and protein decreased, compared with control group; Alisol B can inhibit the effects of C3a(p<0.05)..(3) Indirect immunofluorescence staining showed that the expression of a-SMA increased in HK2 cells, the expression of E-cadherin decreased, while the expression of C3 increased in C3a group in C3a group, compared with control group; Alisol B can inhibit the effects of C3a.(4) The renal pathologic stain of SOR group showed no abnormalities; The damage of interstitium of UUO group was heavier than that of SOR group , The Alisma orientalis can improve these lesions.(5) Immunohistochemistry showed that the expression of a-SMA increased ,while the expression of E-cadherin in tubular epithelial cells decreased in UUO group;Meanwhile,the expression of C3 significantly increased and was inversely correlated with the E-cadherin ,but which was directly correlated with a-SMA.The Alisma orientalis may improve these changes.Conclusion(1) Exogenous C3a induces the expression of C3 and Epithelial-to-Mesenchymal Transition in HK-2; Alisol B Inhibits the expression of C3 induced by C3a and Epithelial-to-Mesenchymal Transition in HK-2.(2) The Alisma orientalis can reduce the expression of C3 in UUO rats,and inhibit Epithelial-to-Mesenchymal Transition in human renal tubular epithelial cells,which play a renoprotective effect.