| Objective :To evaluate the effects of matrine on cell proliferation and expression of intercellular VEGF in RRMECs in vitro and to provide a new clue and theory basis for developing a drug of preventing and curing retinal neovascularization in the future.Methods : RRMECs were primary cultured, the third generation of RRMECs were treated. The effect of Mat on the proliferation of RRMECs was measured by MTT assay. The influence of Mat on the growth of RRMECs was determined by cell counting method in trypan blue staining. AO/EB double fluorescence staining method was used to observe the morphological changes of RRMECs apoptosis after treatment with Mat. RRMECs were divided into 5 groups: the control group, angiostatin (130mg/L) group, Mat (40mg/L) group, high glucose (25mmol/L)group, and Mat(40mg/L)+high glucose(25mmol/L) group. After 48 hour, the localization of VEGF protein was examined by immunocytochemistry. Western-blotting was used to measure the protein level of VEGF.Results :1.We observed the inhibitory effects of Mat on RRMECs through MTT assay, which were exposed to 5mg/L,10mg/L,20mg/L,40mg/L,80mg/L and 160mg/L concentrations for 24, 48, 72 hours respectively. The inhibitory rate were 1.42±1.03%,11.50±1.98%,24.98±2.38%,40.27±2.49%,53.54±2.39%,63.88±3.12% after they were incubated with Mat for 24 hours, they are 4.16±1.14%, 14.40±1.78%, 33.28±1.83%,53.61±1.32%,63.53±1.96%, 73.79±1.12% for 48 hours and 9.83±2.13%, 22.17±4.29%, 42.65±1.05%, 63.17±1.38%, 74.35±2.60%, 87.24±4.59%.for 72 hours. When Mat concentrations exceed 5mg/L, there were significant differences between experimental groups in which the RRMECs were disposed with different concentrations and incubation time of Mat and control group disposed with DMEM (P<0.05), as well as among experimental groups (P<0.05). The inhibitory rates of RRMECs increased with both the raise of the Mat concentrations and the prolongation of time. These results were consistent with the results of cell counting method drawing growth curve in RRMECs. 2. AO/EB double fluorescence staining method indicated that we could observe typical apoptotic cells with fluorescence microscope when Mat whose concentration exceed 5mg/L. 3. Immunohistochemical techniques indicated that the expression of VEGF is in cell membranes and cytoplasm. Compared with the control group, An and Mat decreased the expression of VEGF (P<0.05); Compared with the control group, high glucose increased the expression of VEGF (P<0.05). Mat could decrease the expression as well (P<0.05). These results were consistent with the results of Western-blotting.Conclusions :1. Mat can effectively inhibit the proliferation of RRMECs, the inhibitory rates are increased in both time-dependent and dose-dependent ways by Mat in vitro. 2. Mat can effectively induce the apoptosis of RRMECs in vitro. 3. Immunohistochemistry and Western-blotting analysis, the effects of growth inhibition of Mat on RRMECs are associated with down-regulating expression of VEGF protein , which may be one of important mechanisms to inhibit the cells proliferation. It suggests that Mat may be a potent drug for the treatment of retinal neovascularization.
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