| Gastric cancer is one of the most common malignant tumors in China.Its incidence rate is high,mortality is high,and it seriously endangers human health.Therefore,it is urgent to develop the treatment of gastric cancer.Aesculetin is a kind of coumarin compound extracted from cortex Fraxini,which has anti-inflammatory,antibacterial,anti-oxidation,anti-tumor and other pharmacological effects.In recent years,studies have shown that Aesculetin can inhibit the proliferation,invasion and migration of a variety of tumors,induce apoptosis and other functions,and involve multiple signaling pathways.In this study,SGC-7901 cell line of human gastric cancer was selected as the research object.In previous studies,it was shown that Aesculetin can induce apoptosis through mitochondrial pathway and death receptor pathway.Therefore,the mechanism of Aesculetin induced apoptosis of SGC-7901 cell line of human gastric cancer was discussed in this study,which provided theoretical basis for the mechanism of Aesculetin anti-tumor.Methods:(1)Western blot was used to detect the expression of ER stress-related protein GRP78 and CHOP in human gastric cancer SGC-7901 cells;(2)MTT was used to detect the effect of Aesculetin on the proliferation of SGC-7901 cells;Hoechst 33258 method was used to detect the effect of Aesculetin on SGC-7901 cells apoptosis;(3)H2DCFDA staining was used to detect the effect of Aesculetin on ROS level in SGC-7901 cells;MTT method was used to detect the effect of NAC on the inhibition of SGC-7901 cells proliferation by Aesculetin;Hoechst 33258 method was used to detect the effect of NAC on the apoptosis of SGC-7901 cells induced by Aesculetin;(4)The expression of GRP78,p-PERK,p-eIF2?,ATF-4 and CHOP protein in SGC-7901 cells was detected by Western blot method,and the effect of TUDCA on the proliferation of SGC-7901 cells was detected by MTT method.The effect of TUDCA on apoptosis of SGC-7901 cells induced by Aesculetin was detected by Hoechst 33258 method;(5)The effect of TUDCA on ROS level of SGC-7901 cells induced by Aesculetin was detected by H2 DCFDA staining;the effect of NAC on GRP78 and CHOP protein expression of SGC-7901 cells induced by Aesculetin was detected by Western blot.Results:(1)Western blot was used to detect the expression of GRP78 and CHOP in SGC-7901 cells,suggesting that the balance of GRP78 and CHOP expression was related to the development of SGC-7901 cells.(2)The proliferation of SGC-7901 cells was inhibited by Aesculetin by MTT assay.With the increase of Aesculetin concentration,the cell viability of SGC-7901 cells was lower and dose-dependent.Hoechst 33258 staining was used to observe the morphological changes of the nucleus and cytoplasm of cells treated with Aesculetin 0.28,0.56 and 1.12 mmol/L.it was found that with the increase of Aesculetin concentration,there were obvious morphological characteristics of apoptosis such as karyopyknosis and nuclear fragmentation,which suggested that Aesculetin could play its anti-cancer activity by inducing apoptosis of SGC-7901 cells.(3)The level of ROS in the cells treated with 0.28,0.56 and 1.12 mmol/L Aesculetin was significantly increased and dose-dependent by H2 DCFDA staining.After 1 hour of pretreatment with 100 ?mol/L NAC,the cell viability(67.25±4.28)% in the NAC+Aesculetin group was significantly higher than that in the Aesculetin group(56.36±4.68)%(P<0.05).The cell apoptosis of NAC+Aesculetin group was significantly lower than that of Aesculetin group,indicating that inhibition of ROS can inhibit the apoptosis of Aesculetin,suggesting that Aesculetin induced apoptosis of SGC-7901 cells is closely related to ROS.(4)Through western blot the expression of GRP78,p-PERK,p-eIF2?,ATF-4 and CHOP protein in the cells treated with 0.28,0.56 and 1.12 mmol/L Aesculetin was up-regulated in varying degrees.After 1 hour of pretreatment with 50 ?mol/L TUDCA,the cell viability(58.77±3.46)% of the cells in the group treated with TUDCA+Aesculetin was significantly higher than that in the group treated with Aesculetin(48.43±2.51)%(P<0.05).It can inhibit the inhibitory effect of Aesculetin on the proliferation of SGC-7901 cells.Hoechst 33258 staining showed that the degree of apoptosis in TUDCA+Aesculetin group was significantly lower than that in Aesculetin group,indicating that inhibition of ER stress can inhibit the apoptosis of Aesculetin,suggesting that Aesculetin can induce the apoptosis of SGC-7901 cells through endoplasmic reticulum stress.(5)The ROS level of TUDCA+Aesculetin group was significantly lower than that of Aesculetin group after pretreated with 50 ?mol/L TUDCA for 1 hour by H2 DCFDA staining,indicating that inhibition of ER stress could inhibit the production of ROS induced by Aesculetin;after pretreated with 100 ?mol/L NAC for 1 hour,the ROS level of TUDCA+Aesculetin group was significantly lower than that of Aesculetin group The expression of GRP78 and CHOP protein in NAC+Aesculetin group was lower than that in Aesculetin group,which indicated that inhibition of ROS could also affect ER stress,suggesting that Aesculetin had dual regulatory effects on ROS and ER stress in SGC-7901 cells.Conclusion: GRP78 and CHOP ER stress-related proteins are expressed in SGC-7901 cells,indicating that GRP78 and CHOP ER stress-related proteins are related to the development of SGC-7901 cells.Aesculetin can inhibit the proliferation of SGC-7901 cells through oxidative stress and ER stress,promote the apoptosis of SGC-7901 cells,and ER stress can modulate the growth of SGC-7901 cells ROS level in SGC-7901 cells,on the contrary,ROS production can also induce endoplasmic reticulum stress,suggesting that Aesculetin induced apoptosis of SGC-7901 cells may be related to the interaction between oxidative stress and endoplasmic reticulum stress. |
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