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Objective To investigate the effects of bevacizumab on cell morphology,apoptosis rate and apoptosis-related factors in human retinal pigment epitheliumcells.Methods Human retinal pigment epithelial cells (ARPE-19) were cultured in DMEM/F1 2 medium containing 0.25 g · L-1 bevacizumab and divided into 1-week dosing group and 2-week dosing group according to the different incubation time,respectively.Meanwhile,the control group was set up.Then,the cell morphology and apoptosis of each group were observed by light microscope and flow cytometry,accordingly.The expression levels of apoptosis-promoting genes P53,TP53INP1,Bax and apoptosisinducible gene Bcl-2 mRNA and protein were detected by RT-PCR and Western blot,respectively.Results Light microscopic observation revealed that the cells in the 1-week control group and the 2-week control group showed typical epithelial cell morphology,and were in stone-like,single-layer adherent-like growth.Compared with the control group,part of the cell morphology after bevacizumab treatment changed slightly,and the cells became rounded,the cell body was elongated and the boundary was poor.The apoptotic rates of the 1-week control group,l-week dosing group,2-weekcontrol group and 2-week dosing group were (5.57 ± 1.46) ％,(6.39 ± 1.25) ％,(6.88 ± 1.10)％ and (13.34 ± 1.94)％,respectively,and there was no significant difference in the apoptotic rate between 1-week control group,1-week dosing group and 2-week control group (both P ＞ 0.05),but the apoptotic rate of the 2-week dosing group was significantly higher than that of the 2-week control group,the 1-week dosing group and 1-week control group,and the differences were statistically significant (all P ＜0.01).RT-PCR and Western blot results showed that compared with the 1-week control group,the expression of P53,TP53INP1 and Bax mRNA was up-regulated in 1-week dosing group,but the expression of Bcl-2 mRNA was down-regulated,and the differences were statistically significant (all P ＜ 0.05).The expression levels of P53,TP53INP1 and Bax mRNA in 2-week dosing group were higher than those in 2-week control group and 1-week dosing group,while Bcl-2 mRNA expression was down-regulated,and the differences were statistically significant (all P ＜ 0.05).There was no significant difference in the expression of the above factors in 1-week control group and 2-week control group(all P ＞ 0.05).The protein expressions of above factors were similar to their mRNA expression.Conclusion Bevacizumab can alter the morphology of ARPE-19 ceils,increase the apoptotic rate and up-regulate the expression of apoptosis-promoting factors,but down-regulate the expression of apoptosis-suppression factors in ARPE-19 cells,which may be the reason for the loss of RPE layer in the macula after anti-VEGF therapy.
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Objective To investigate the effect of procyanidins (PC) on cell apoptosis and cytochrome C releasing in human trabecular meshwork cells (HTMC) under oxidative stress induced by H2 O2.Methods HTMC were cultured and then divided randomly into 5 groups.Normal cultured HTMC served as normal group without treatment,and normal cultured HTMC in control group were treated with 500 μrnol · L-1 H2O2 for 1 h,as well as normal cells in treatment groups were treated with 500 imol · L-1 H2 O2 for 1 h combined with different concentrations of PC (0.02 g · L-1,0.05 g · L-1,0.10 g · L-1).Then flow cytometry and Western blot were applied to detect the cell apoptosis rate and the release of cytochrome C (CytC) respectively.Results Compared with the normal group,the apoptotic rates of the control group and the PC treatment groups were significantly increased,and the difference was statistically significant (all P ＜ 0.01).Compared with the control group,the apoptotic rates of the 3 PC treatment groups were decreased,and the differences were statistically significant (all P ＜ 0.01).Moreover,the apoptotic rate in 0.02 g · L-1,0.05 g · L-1,0.10 g · L-1 PC treatment groups was decreased as PC concentration increased,and the differences were statistically significant (all P ＜ 0.01).Furthermore,compared with the normal group,the release of cytochrome C in the control group,0.02 g · L-1 and 0.05 g · L-1 PC treatment group enhanced,and the differences were statistically significant (all P ＜ 0.01),but there was no significant difference between 0.01 g · L-1 PC treatment group and the normal group (P ＞ 0.05).Compared with the control group,the release of cytochrome C in the 3 PC treatment group was attenuated with significant differences (all P ＜0.01).Meanwhile,the release of cytochrome C was decreased in the 3 PC treatment groups with a concentration-dependent manner,and the pairwise differences were statistically significant (all P ＜ 0.01).Conclusion Exogenous PC can decrease the apoptotic rate of HTMC under oxidative stress and reduce the release of cytochrome C,as well as it also has an antioxidant effect in a certain concentration-dependent manner.
ABSTRACT
Objective To investigate the effect of procyanidins (PC) on cell apoptosis and cytochrome C releasing in human trabecular meshwork cells (HTMC) under oxidative stress induced by H2 O2.Methods HTMC were cultured and then divided randomly into 5 groups.Normal cultured HTMC served as normal group without treatment,and normal cultured HTMC in control group were treated with 500 μrnol · L-1 H2O2 for 1 h,as well as normal cells in treatment groups were treated with 500 imol · L-1 H2 O2 for 1 h combined with different concentrations of PC (0.02 g · L-1,0.05 g · L-1,0.10 g · L-1).Then flow cytometry and Western blot were applied to detect the cell apoptosis rate and the release of cytochrome C (CytC) respectively.Results Compared with the normal group,the apoptotic rates of the control group and the PC treatment groups were significantly increased,and the difference was statistically significant (all P ＜ 0.01).Compared with the control group,the apoptotic rates of the 3 PC treatment groups were decreased,and the differences were statistically significant (all P ＜ 0.01).Moreover,the apoptotic rate in 0.02 g · L-1,0.05 g · L-1,0.10 g · L-1 PC treatment groups was decreased as PC concentration increased,and the differences were statistically significant (all P ＜ 0.01).Furthermore,compared with the normal group,the release of cytochrome C in the control group,0.02 g · L-1 and 0.05 g · L-1 PC treatment group enhanced,and the differences were statistically significant (all P ＜ 0.01),but there was no significant difference between 0.01 g · L-1 PC treatment group and the normal group (P ＞ 0.05).Compared with the control group,the release of cytochrome C in the 3 PC treatment group was attenuated with significant differences (all P ＜0.01).Meanwhile,the release of cytochrome C was decreased in the 3 PC treatment groups with a concentration-dependent manner,and the pairwise differences were statistically significant (all P ＜ 0.01).Conclusion Exogenous PC can decrease the apoptotic rate of HTMC under oxidative stress and reduce the release of cytochrome C,as well as it also has an antioxidant effect in a certain concentration-dependent manner.
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Objective To explore the protective effect of Zhenbao pill on acute spinal cord injury of rat and its possible mechanisms.Methods 64 Wistar rats were randomly divided into a injury group and a drug group, with 32 rats in each group, A T10 acute incomplete spinal cord injury model was produced by using modified Allen technique in the two groups,Zhenbao pill mixed suspension of 0.6 g/kg was given by gavage method daily in the drug group 30 min after moding, the same amount of saline was given in the injury group. Behavior detection were conducted in the first day, third day, and the seventh day after modeling to each 8 rats of the two groups. Spinal cord specimens was got centered about the damage points after rats were executed. histopathological changes were observed under HE staining, the MDA content were determined by thiobarbituric acid spectrophotometer and the apoptotic cells were marked by TUNEL method.Results After 1, 3, 7 d, compared with the injury group, the MDA level in the rat spinal cord tissue in the drug group (15.12 ± 1.27 nmol/mgvs.19.71 ± 1.29 nmol/mg, 20.42 ± 1.33 nmol/mgvs.24.65 ±1.36 nmol/mg, 10.46±1.49 nmol/mg vs.15.46 ± 1.44 nmol/mg) decreased(P<0.05); the apoptosis rate of drug groups (19.61% ± 1.49%vs.26.61% ±1.52%, 21.49% ± 1.37% vs.32.37% ± 1.24%, 13.04% ± 1.30%vs.18.35% ± 1.27%)decreased (P<0.05); the BBB score of drug groups (6.52 ± 1.27vs. 2.63 ± 1.27), (12.68 ± 1.32 vs.6.17 ± 1.34), (15.47 ± 1.27 vs.11.57 ± 1.29) was higher than the injury group (P<0.05). Conclusion Zhenbao pill can significantly improve the histopathological examination,reduce the MDA content in the injured spinal cord specimen of the rat and the rate of neuronal apoptosis, which may promote recovery of neurologic function in the rat suffered from ASCI.