| Objective: To explore the effect of Smilax China L.on the endoplasmic reticulum stress-autophagy of human hepatocellular carcinoma SMMC-7721 cells,and to reveal the mechanism of action of Smilax China L.on anti-liver cancer.Methods: Part 1: Divide SMMC-7721 cells into Control group,JGT-L group,JGT-M group,JGT-H group,and give blank serum and different concentrations(0.5g/m L,1g/m L,2g/m L))The Smilax China L.-containing serum intervened for 48 hours.(1)The survival rate of SMMC-7721 cells in each group was detected by CCK-8 method at 12 h,24h and 48 h.(2)Flow cytometry was used to detect the cell cycle of SMMC-7721 cells in each group.(3)Observe the ultrastructure of SMMC-7721 cells in each group by transmission electron microscope.(4)The q RT-PCR method was used to detect the expression of e IF2α and ATF4 m RNA of SMMC-7721 cells in each group.(5)Western blot was used to detect the expression of PERK,p-PERK,e IF2α,ATF4,and CHOP protein in SMMC-7721 cells in each group.Part 2:Dithiothreitol(DTT)was used to intervene in SMMC-721 cells for 24 hours to construct an endoplasmic reticulum stress-autophagy cell model.SMMC-7721 cells are randomly divided into Control group(blank drug-containing serum),DTT group(500μM DTT),DTT+JGT group(500μM DTT+2g/ml Smilax China L.-containing sera),JGT group(2g/ml Smilax China L.-containing sera)Drug serum),cells were collected after 12 hours of culture in each group.(1)The CCK-8 method was used to detect the proliferation ability of SMMC-7721 cells in each group.(2)Observe the endoplasmic reticulum and autophagosomes of SMMC-7721 cells in each group by transmission electron microscope.(3)The q RT-PCR method was used to detect the expression of TRAF2 and Beclin1 m RNA in SMMC-7721 cells in each group.Results: Part Ⅰ:(1)Comparison of the proliferation of SMMC-7721 cells in each group: Compared with the Control group at the same time point,the various concentrations of the Smilax China L.drug group acted on SMMC-7721 cells after 12 h,24h,and 48 h.As the concentration of Smilax China L.-containing serum increased,the survival rate of SMMC-7721 cells also decreased significantly in turn(all p<0.05).The cell survival rate of the JGT-L group,JGT-M group,and JGT-H group gradually decreased.When comparing the groups,p was <0.05.After 12 hours,24 hours,and 48 hours of intervention in the SMTC-7721 cells of various concentrations of the Smilax China L.drug group,their IC50 of inhibitory concentrations were 1.369 g/L,1.007 g/L,and 0.895 g/L,respectively.(2)Comparison of SMMC-7721 cell cycle phase cells in each group: Compared with the Control group,the number of cells in the G0/G1 phase in the JGT-M and JGT-H groups increased significantly(p<0.05),JGT-H The number of cells in the S phase of the group was significantly reduced(p<0.05),and the cells in the G2/M phase of the JGT-L and JGT-M groups were significantly reduced(p<0.05).(3)Ultrastructure of SMMC-7721 cells in each group: the nucleus of the Control group was oval,the nuclear membrane was intact,the endoplasmic reticulum lumen was slightly expanded in the cytoplasm,and the granular endoplasmic reticulum was slightly degranulated.A large number of organelles can be seen in various concentrations of the Smilax China L.drug group.The organelle density increases,the nucleus shrinks,and the chromatin becomes dense.It is distributed in the nucleus and the inner nuclear membrane in the form of particles or blocks.Occasionally,autophagosomes are seen.Among them,some vacuoles can be seen in the cytoplasm of the JGT-L group and the JGT-M group,a small amount of granular endoplasmic reticulum is loosely dispersed in the cytoplasm,and a small amount of granular endoplasmic reticulum is degranulated;the rough endoplasmic reticulum is visible in the cytoplasm of the JGT-H group It is relatively neatly arranged near the cell nucleus,and the endoplasmic reticulum of the granular type is slightly degranulated.(4)Comparison of the expression of e IF2α and ATF4 m RNA in SMMC-7721 cells of each group: Compared with the Control group,the m RNA expression of e IF2α in JGT-M and JGT-H groups was significantly reduced(p<0.001),and the expression of ATF4 m RNA in JGT-H group Significantly reduced(p<0.001).(5)Comparison of PERK,p-PERK,e IF2α,ATF4,and CHOP protein expression of SMMC-7721 cells in each group: Compared with the Control group,the expression of p-PERK protein in the JGT-H group was significantly reduced(p<0.05),each The expression of e IF2α protein was significantly reduced in the concentration of Smilax China L.drug group(p<0.05),and the expression of ATF4 in the JGT-H group was significantly reduced(p<0.05).The expression of CHOP protein in SMMC-7721 cells in each group was not statistically significant.Part II:(1)Comparison of the proliferation of SMMC-7721 cells in each group: Compared with the Control group,the cell proliferation ability of the DTT,DTT+JGT,and JGT groups all decreased(p<0.05);compared with the DTT group,DTT+JGT The cell proliferation ability of the group increased(p<0.05),and the cell proliferation ability of the JGT group decreased(p<0.05);compared with the DTT+JGT group,the cell proliferation ability of the JGT group decreased(p<0.05).(2)Endoplasmic reticulum and autophagosomes of SMMC-7721 cells in each group: SMMC-7721 cells in the Control group showed a slight expansion of the endoplasmic reticulum lumen,a small amount of granular endoplasmic reticulum was degranulated,and some autophagosomes were visible in the cytoplasm;In the DTT group,SMMC-7721 cells had reduced organelles,and a large number of autophagosomes were seen in the cytoplasm;the DTT+JGT group showed that the endoplasmic reticulum lumen of the cells was slightly expanded,the granular endoplasmic reticulum was degranulated,and a small amount of autophagosomes was seen in the cytoplasm.In the endoplasmic reticulum lumen of the JGT group,the expansion was not obvious,and most of the granular endoplasmic reticulum and autophagosome were occasionally found in the cytoplasm.(3)Comparison of the expression of TRAF2 and Beclin1 m RNA in SMMC-7721 cells of each group: Compared with the Control group,the expressions of TRAF2 and Beclin1 m RNA in the DTT and DTT+JGT groups were increased(all p<0.05),and the TRAF2 and Beclin1 m RNA in the JGT group Expressions were all decreased(all p<0.05);compared with the DTT group,the expressions of TRAF2 and Beclin1 m RNA in the DTT+JGT and JGT groups were reduced(all p<0.05);compared with the DTT+JGT group,the expressions of TRAF2 and Beclin1 m RNA in the JGT group Expression decreased(all p<0.05).Conclusion: Smilax China L.can inhibit the proliferation of SMMC-7721 and block cells in the G1 phase.The mechanism may be related to its inhibition of the PERK-ATF4 pathway in response to endoplasmic reticulum stress unfolded protein reaction,which in turn inhibits endoplasmic reticulum stress-autophagy. |
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