| Ischemic stroke is a sudden onset of cerebral blood flow and then cause ischemia and hypoxia,leading to localized brain tissue ischemic necrosis or brain softening of the disease.If cerebral ischemic patients do not receive timely treatment,severe cases will suffer from visual impairment,loss of language,paralysis and confusion,such as varying degrees of dysfunction,and even lead to death,Ischemic stroke is a serious hazard to human health.Chinese medicine has a unique advantage on prevention and treatment of ischemic stroke,and the medical profession has been recognized ischemic stroke is the dominant disease of traditional Chinese medicine.The research group has accumulated experience in the study of ischemic stroke,found that Qingkailing,refined Qingkailing have a good effect on ischemic stroke model rats,which gardenia,geniposide as the main ingredient played an important role effect.In the pathogenesis of ischemic stroke,in addition to inflammation,local congestion stimulation,nerve cell damage,apoptosis is a very important part of the disease.And the role of Endoplasmic reticulum stress and autophagy in the study of neuronal injury get more and more attention.Endoplasmic reticulum stress and autophagy are independent and closely linked in the pathogenesis of ischemic stroke At present,most studies suggest that endoplasmic reticulum stress can promote autophagy,in turn,autophagy can weaken the endoplasmic reticulum stress.This may be related to autophagy by removing excess waste protein and thus weakening the endoplasmic reticulum stress.There is a hypothesis that autophagy by weakening the excessive endoplasmic reticulum stress to combat neuronal apoptosis.In this study,we studied the protective effect of gardenia and its representative component of geniposide on neuronal damage induced by energy barrier at the cellular level.And to explore the mechanism of geniposide on cell apoptosis,endoplasmic reticulum stress and autophagy,hoping to provide more scientific basis for the neuroprotective effect of gardenia and geniposide.1 Purpose1.1 Detect the toxicity of gardenia and geniposide in SH-SY5Y cells and the dose range.1.2 To clear the gardenia,geniposide protective effect on normal cells and glucose-deprivation model of SH-SY5Y cells.1.3 To investigate whether the neuroprotective effect of geniposide is associated with anti-apoptotic,interfering with endoplasmic reticulum stress and autophagy.2 method2.1 Establish SH-SY5Y Sugar and oxygen deprivation reperfusion model.2.2 CCK-8 was used to detect the cell survival rate.2.3 JC-1 staining was used to detect apoptosis.The expression of caspase12 were detected by Western blot.2.4 The expression of Bip/GRP78.CHOP were detected by Western blot.2.5 Acridine orange,MDC fluorescent dye on the cells were stained,a preliminary understanding of the neuroprotective effect of geniposide is related to autophagy.The expression of LC3 ? and P62 protein was detected by indirect immunofluorescence assay and Western blot.3 Results3.1 Detection of gardenia,geniposide drug toxicity.3.1.1 The results of CCK8 showed that the concentration of 0.1 ?g/mL and ?g/mL of gardenia increased the activity of normal cells and promoted the growth of normal cells(P<0.05).The highest concentration of 2000?g/mL of Gardenia water extract on the normal cells showed inhibitory effect(P<0.001).3.1.2 The results of CCK-8 showed that there was no significant difference between the concentration of geniposide and the normal group.3.2 Effect on Cell of Sugar and Oxygen Deprivation-Recipe Reoxygenation Model.3.2.1 Effects of Gardenia Extract and Geniposide on OGD12h-Rep2h Model Cells? 10?g/mL,50?g/mL,100?g/mL,500?g/mL and 1000?g/mL Gardenia extract were significantly higher than those in the model group(P<0.05).And the protective effect was dose-dependent relationship with the concentration of the drug.The higher the concentration of gardenia,the more obvious the protective effect.?There was no significant difference in cell viability between 1?g/mL geniposide and 10?g/mL geniposide,and the cell viability of 100?g/mL,500?g/mL and 1000?g/mL geniposide group was significantly different from that of model group(P<0.05).3.2.2 Effect of Geniposide on Model Cells of OGD-Rep at Different Time.?OGD6h-Rep2h,the degree of cell damage is very light.1?g/mL,10?g/mL,100?g/mL geniposide group compared with the model group,the cell viability was significantly increased(P<0.05).500 ?g/mL geniposide group,1000 ?g/mL geniposide group and positive drug edaravone group were not significantly enhanced compared with the model group.?OGD9h-Rep2h,the degree of injury than 6h weight,positive drug edaravone still did not show a protective effect,1?g/mL geniposide group disappeared,10?g/mL geniposide group,100?g/mL geniposide group,500?g/mL geniposide group,1000?g/mL geniposide group compared with the model group was significantly enhanced(P<0.05),and with the increase of geniposide concentration,the protective effect of SH-SY5Y was more obvious.?OGD11h-Rep2h,the degree of injury than 9h weight,positive drug edaravone show a protective effect,1?g/mL geniposide group disappeared,10?g/mL geniposide group,100?g/mL geniposide group,500?g/mL geniposide group,1000?g/mL geniposide group compared with the model group was significantly enhanced(P<0.05),and with the increase of geniposide concentration,the protective effect of SH-SY5Y was more obvious.3.3 Comparison of the efficacy of gardenia water extract and geniposide-CCK-8 test showed that the protective effect of gardenia at 500 ?g/mL was stronger than that of 83.5 ?g/mL of geniposide.3.4 Research on mechanisms3.4.1 JC-1 staining showed that geniposide could reduce OGD12h-Rep2h-induced apoptosis.Western blot results showed that the expression of caspase12 in the model group was significantly higher than that in the normal group(P<0.001),while that in 500?g/ml geniposide group,100?g/ml geniposide group,10?g/ml geniposide group was lower than that in the model group(P<0.01).3.4.2 Endoplasmic reticulum stress.Western blot analysis showed that the expression of CHOP and GRP78/Bip in the endoplasmic reticulum were significantly higher than those in the normal group(P<0.001),10?g/ml geniposide group,100?g/ml geniposide group,500?g/M1 of geniposide CHOP and GRP78/Bip were lower than those in model group(P<0.01).3.4,3 Autophagy Autophagic staining:a,acridine orange staining results,the normal group showed green fluorescence signal,did not show significant autophagy,model group and 10?g/ml,100 ?g/ml,500 ?g/ml geniposide group showed significant red fluorescence.The model group red fluorescence was obvious than geniposide group,and the red fluorescence of the 500 ?g/ml geniposide group was the least,and the difference between the middle 10?g/ml and 100 ?g/ml,geniposide group was not obvious.MDC staining results:the normal group of fluorescence is weak than the other groups,and the fluorescence showed a state of dispersion.There were many peaks in the nucleus of the model group,showing strong fluorescence.Geniposide administration group was weaker than model group.Indirect immunofluorescence assay LC311 results:There was a small amount of green fluorescence in the cytoplasm of normal cells.The green fluorescence in the cytoplasm of the model group was higher than that in the normal group,and the fluorescence signal was stronger than that of the normal group.The fluorescence signal of 500?g/ml geniposide group was weaker than that of model group,and the fluorescence signal of 10?g/ml geniposide group was weaker than that of model group.Indirect immunofluorescence assay P62 results:There was a significant green fluorescence signal in the normal cells.The green fluorescence in the cytoplasm of the model group was lower than that in the normal group.The green fluorescence signal of 500?g/ml and 10?g/ml geniposide group was higher than that of the model group The fluorescence signal of 100?g/ml geniposide group was not obvious compared with the model group.Western blot results showed that the expression of autophagy-related protein LC3? in model group was higher than that in normal group,and the expression of LC3? in the 500?g/ml geniposide group was lower than that of model group,but there was no statistically significant difference between the model group and the 10?g/ml geniposide group,1000?g/ml geniposide group.The expression of autophagy-related protein P62 in model group was lower than that in normal group,and the the expression of P62 in the 500?g/ml geniposide group was higher than that of model group,but there was no statistically significant difference between the model group and the 10?g/ml geniposide group,1000?g/ml geniposide group.4 Conclusion4.1 Gardenia extract has a growth promoting effect on SH-SY5Y cells.4.2 Gardenia extract and geniposide had protective effect on SH-SY5Y cells induced by OGD-Rep,and the protective effect of Gardenia japonica was stronger than that of geniposide.4.3 The protective effect of geniposide on SH-SY5Y cells damaged by OGD-Rep may be achieved by inhibiting the induction of endoplasmic reticulum stress and inhibiting apoptosis.4.4 Geniposide can reduce intracellular autophagy levels in SH-SY5Y cells damaged by OGD-Rep. |
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