Influence Of Mas Gene Silencing On The Effect Of Angiotensin-(1-7) Antagonize The Renal Interstitial Fibroblast Activating By Angiotensinâ…¡

Objective: In the first step of our experiment, we selectedthe most effective sequence of Mas small interfering RNA (SiRNA)already. Now in order to explore the influence of Mas gene silencing onthe effect of angiotensin-(1-7)[ang-(1-7)] that antagonize angiotensinⅡ(Ang Ⅱ) which induced rat renal interstitial fibroblast (NRK-49F)activation, we have the research. Methods:NRK-49F are maintained andsub-cultured in vitro. Research is performed after the cells′confluencegrew up to70-80%and then been prepared for1×105/ml suspension withDMEM-F12in six-well plates. In the first step of our experiment, wedesigned and synthesized three different sequences of Mas SiRNA andtransient transfected of NRK-49F by means of HiPerFect TransfectionReagent. Semi-quantitative PCR(RT-PCR) was used to detect the mRNAexpression of Mas gene. Western blotting was used to detect the proteinexpression of Mas gene. According to the results of RT-PCR and Westernblotting, we found that Mas SiRNA could inhibit the expression of Masgene effectively and selected the most effective Mas SiRNA sequencealready. Now in order to explore the influence of Mas gene silencing onthe effect of ang-(1-7) that antagonize AngⅡwhich induced rat renal interstitial fibroblast activation, we use Mas SiRNA that selected alreadyto transient transfect the NRK-49F. After cultured for48h, cells isdivided into6groups:①normal control group(NG): the cells aremaintained in the DMEM-F12medium supplemented with10%FBSand1%penicillin-streptomycin.②A ngⅡ group: the cells are maintainedin the DMEM-F12medium supplemented with10%FBS and theangiotensinⅡ that the final concentration was10-6mol/L.③A ng-(1-7)group: the cells are maintained in the DMEM-F12medium supplementedwith10%FBS and the angiotensin-(1-7) that the final concentration was10-5mol/L.④AngⅡ+Ang-(1-7) group: the cells are maintained in theDMEM-F12medium supplemented with10%FBS,10-6mol/LangiotensinⅡ and10-5mol/L angiotensin-(1-7).⑤A ngⅡ+Ang-(1-7)+negative SiRNA group (A+SiRNA-CG): After been transient transfectedby the negative SiRNA for48h, the cells are maintained in theDMEM-F12medium supplemented with10%FBS,10-6mol/LangiotensinⅡ and10-5mol/L angiotensin-(1-7).⑥A ngⅡ+Ang-(1-7)+Mas SiRNA (A+MasSiRNA): After been transient transfected by theMas SiRNA SiRNA for48h, the cells are maintained in the DMEM-F12medium supplemented with10%FBS,10-6mol/L angiotensinⅡ and10-5mol/L angiotensin-(1-7). The cells above are cultured for72h.Thenthe expression of α-smooth muscle actin (α-SMA) is detected byimmunocytochemistry method. The content of CollagenⅠ(ColⅠ) in the cultured supernatant is measured by ELISA. Results:①the resultsofimmunocytochemistry: After cultured with AngⅡand Ang-(1-7) for72h,the cells of Mas SiRNA transfection group could see hypertrophy and theratio of cytoplasm is increasing. There are a amount of boundary clearbrown granules in the cytoplasm and is similar to the cells of AngⅡgroup. And compared with the non-transfection group and negativeSiRNA transfection group, the expression of α-SMA increasedsignificantly in the cell after analysis of data (P<0.05). There are nosignificant difference between AngⅡ+Ang-(1-7) group and AngⅡ+Ang-(1-7)+negative SiRNA group (P>0.05). In control group, there isbasic expressions of α-SMA.②ELISA: After cultured with AngⅡ andAng-(1-7) for72h, the secretion of ColⅠ is rised significantly in thecultured supernatant of Mas SiRNA transfection group than the non-transfection group and negative SiRNA transfection group (P <0.05). Thecontent of ColⅠin the AngⅡ+Ang-(1-7) group and A+SiRNA-CG areno significant difference (P>0.05). There is basic secretion of Col Ⅰincontrol group and Ang-(1-7) group. Conclution: After the sequence ofMas SiRNA inhibited the expression of Mas gene effectively, thefunction of angiotensin-(1-7) on antagonize the renal interstitial fibroblastactivating by angiotensinⅡ is eliminated significantly.