| Purpose:(1)Discuss the action mechanism of Angiotensin Ⅱ(Ang Ⅱ)in Diabetic nephropathy(DN)through external cell experiment,definite the multiplication of Ang Ⅱ to human glomerular mesangial cells(HMC)and intervention effect and mechanism of Ang Ⅱ to Saponin of Litchi Seed(SLD).(2)Definite the significance of inflammatory interleukin-1β(IL-1β)and interleukin-6(IL-6)in the development of DN.(3)Research the position and function of suppressor of cytokine signaling-1(SOCS-1)in DN,definite the expression of SOCS-1 can reduce the inflammatory reaction of DN.Method:(1)Divide HMC cell into 5 groups:(1)Normal group: culture cell normally;(2)Ang Ⅱ 10-6mol/ml group;(3)Ang Ⅱ 10-7mol/ml group;(4)Ang Ⅱ 10-8 mol/ml group;(5)Blank zero group: no cell.(2)Screen the effective action concentration of Ang Ⅱ through MTT colorimetric method at first,then apply SLD intervention in different concentrations(5mg/ml,10mg/ml,20mg/ml);collect supernate and cell of each group after intervention for 48 hours.(3)Test the HMC cell proliferation with MTT colorimetric method;detect the content of cell supernatant IL-1β and IL-6 by ELISA method;detect the expression of signaling protein SOCS-1 with western blot method.Result:(1)The MTT detection results show that the absorbance value(OD value)(P<0.05)is increased obviously after Ang Ⅱ acted on HMC cell.The function of 10-6 mol/ml Ang Ⅱ is highest in 48 hours(P<0.01).(2)The MTT detection results show that high,medium and low dose group of SLD can obviously restrain the multiplication of HMC cell(P<0.01),high dose group of SLD has the highest inhibition rate(55.43%).(3)ELISA detection results show that all dose groups of SLD can restrain the level of IL-1β and IL-6(P<0.01 or P<0.05)compared with Ang Ⅱ group.(4)Western Blot experiment results show that the expression quantity of SOCS-1 protein is increased and higher than that of normal group obviously(P*<0.01).Compared with Ang Ⅱ group,expression quantity of protein is reduced significantly(P*<0.01)while still higher than normal group.Conclusion:(1)Ang Ⅱ can induce HMC cell proliferation and the effect of 10-6 mol/ml is best in 48 hours.(2)Inhibition ratio is increased with the increasing of SLD concentration as well as the extension of time.It has proved that inhibiting effect of SLD to HMC cell proliferation is depended on time and concentration.(3)SLD can reduce the content of cytokines IL-6 and IL-1β secreted by HMC cell obviously and decrease the secretion of extracelluar matrix(ECM)to slow down the sclerosis process of glomerulus.(4)Ang Ⅱ can stimulate the expression of inflammatory factor of HMC cell through SOCS-1 signal path;while SLD can restrain the expression of HMC cell protein to provide a new idea for researching diabetic nephropathy. |
