清肝利水方调控TRPC6/Ca^(2+)通路抑制慢性青光眼RGC凋亡的研究  

Research of Qinggan Lishui Formula on regulating TRPC6/Ca^(2+)signaling pathway to inhibit the apoptosis of RGC in chronic glaucoma

作　　者：陈梦婷 董志国[1] 张殷建[1] 田源 CHEN Mengting;DONG Zhiguo;ZHANG Yinjian;TIAN Yuan(Longhua Hospital,Shanghai University of Traditional Chinese Medicine,Shanghai 200032,China)

机构地区：[1]上海中医药大学附属龙华医院,上海200032

出　　处：《中国中医眼科杂志》2022年第1期5-11,共7页China Journal of Chinese Ophthalmology

基　　金：国家自然科学基金项目(81804147)。

摘　　要：目的观察清肝利水方(QGLS)在慢性高眼压大鼠视网膜神经节细胞(RGC)凋亡中的抑制效果,并探讨其作用机制与瞬时受体电位阳离子通道6(TRPC6)/Ca^(2+)信号通路的关系。方法制备大鼠QGLS含药血清,进行细胞毒性试验,确定合适的干预浓度。用CoCl_(2)和无糖DMEM建立RGC-5细胞缺氧缺糖-复氧复糖损伤模型,CCK-8检测正常对照组(CG)、模型组(MG)和低、中、高浓度清肝利水方含药血清组(LQGLS、MQGLS、HQGLS)的细胞增殖力,细胞凋亡试验检测细胞凋亡水平,Western Blot检测各组TRPC6、B细胞淋巴瘤-2(Bcl-2)、Bcl-2相关X(Bax)和半胱氨酸蛋白酶-3(Caspase-3)蛋白表达,Fluo-3 AM荧光探针检测细胞内钙离子浓度。结果(1)实验条件筛选:筛选出QGLS含药血清干预浓度为5%,CoCl_(2)造模浓度与时间为600μM处理24 h。(2)细胞存活率:LQGLS、MQGLS可显著提高缺氧缺糖-复氧复糖损伤的细胞存活率,与MG比较有统计学意义(t_(LQGLS)=25.401,t_(MQGLS)=11.805,均P=0.000)。(3)TRPC6表达:MQGLS、HQGLS较MG显著降低,差异均有统计学意义(t_(MQGLS)=7.365,t_(HQGLS)=9.075,均P=0.000)。(4)凋亡因子:①Caspase-3表达,3个QGLS剂量组较MG均显著下降,差异均有统计学意义(t_(LQGLS)=4.816,P=0.001;t_(MQGLS)=35.131,P=0.000;t_(HQGLS)=28.227,P=0.000);②Bax表达,3个QGLS剂量组较MG均显著下降,差异均有统计学意义(t_(LQGLS)=14.912,t_(MQGLS)=10.194,t_(HQGLS)=12.838,均P=0.000);③Bcl-2表达,3个QGLS剂量组较MG均显著升高,差异均有统计学意义(t_(LQGLS)=8.871,t_(MQGLS)=12.729,t_(HQGLS)=9.298,均P=0.000)。(5)细胞内Ca^(2+)荧光强度:MG及3个QGLS剂量组较CG均显著升高,差异具有统计学意义(t_(MG)=13.960,P=0.000;t_(LQGLS)=5.889,P=0.001;t_(MQGLS)=7.877,P=0.000;t_(HQGLS)=17.815,P=0.000)。LQGLS、MQGLS较MG细胞内Ca^(2+)浓度显著下降,差异具有统计学意义(t_(LQGLS)=7.881,t_(MQGLS)=6.204,均P=0.000),HQGLS较MG差异无统计学意义(P>0.05)。结论QGLS可降低RGC-5中Caspase-3、Bax表达,提OBJECTIVE To observe the inhibitory effect of Qinggan Lishui Formula(QGLS)on retinal ganglion cell(RGC)apoptosis in rats with chronic high intraocular pressure,and to explore the relationship between its mechanism and transient receptor potential channel 6(TRPC6)/Ca^(2+)signaling pathway.METHODS The serum containing QGLS and the cytotoxicity test was carried out to determine the appropriate intervention concentration.Cobalt chloride(CoCl_(2))and sugar-free DMEM were used to establish a hypoxia-hypoglycemia and reperfusion injury model of RGC-5 cells.CCK-8 was used to detect the proliferation of RGC-5 cells in control group(CG),model group(MG)and QGLS containing serum of different concentration groups(LQGLS,MQGLS,HQGLS).The level of apoptosis was detected by cell apoptosis kit.The protein expressions of TRPC6,B-cell lymphoma-2(Bcl-2),Bcl-2 Associated X(Bax)and Caspase-3 were detected by Western blot.The intracellular calcium concentration was detected by Fluo-3 AM.RESULTS(1)Screening of experimental conditions:The intervention concentration of serum containing QGLS was 5%,and the concentration and time of cobalt chloride modeling were 600μM for 24 h.(2)Cell survival rate:Compared with MG,LQGLS,MQGLS groups significantly improved the cell survival rate of hypoxia-hypoglycemia and reperfusion injury model,and the difference was statistically significant(t_(LQGLS)=25.401,t_(MQGLS)=11.805,all P=0.00).(3)TRPC6 expression:Compared with the MG group,the MQGLS and HQGLS groups were significantly lower(t_(MQGLS)=7.365,t_(HQGLS)=9.075,all P=0.000).(4)Apoptotic factor:①Caspase-3 expression:Compared with the MG group,results of the LQGLS,MQGLS and HQGLS groups were significantly lower,and the difference was statistically significant(t_(LQGLS)=4.816,P=0.001;t_(MQGLS)=35.131,P=0.000;t_(HQGLS)=28.227,P=0.000);②Bax expression:Compared with the MG group,results of the LQGLS,MQGLS and HQGLS groups were significantly lower and the difference was statistically significant(t_(LQGLS)=14.912,t_(MQGLS)=10.194,t_(HQGLS)=12.838,all P=

关 键 词：清肝利水方 慢性高眼压 青光眼 视网膜神经节细胞 TRPC6 凋亡蛋白 

分 类 号：R285.5[医药卫生—中药学]