Mechanisms Of Hydroxysafflor Yellow A Improves Oxidative Stress Induced By High Glucose In Pancreatic β-cells

BackgroundDiabetes mellitus is a group of metabolic diseases,which plagues human health,characterized by hyperglycemia caused by defects in insulin secretion and its biological dysfunction.It is often clinically manifested by polydipsia,polyphagia,polyuria and weight loss.Excessive amount of ROS induced by high glucose disrupts the balance of oxidant production and antioxidant defenses of pancreatic β cells,which results in apoptosis and impaired GSIS function.Meanwhile,low expression of the principal antioxidant enzymes and poor DNA capacity against oxidative stress make isletβcells more vulnerable to oxidative stress than other tissues.Protecting the viability and function of islet β cells under oxidative stress is of great significance for diabetic prevention and treatmentSafflower is a traditional Chinese medicine,and it has a history of more than 2,000 years.Safflower yellow A is the most important water-soluble component of safflower.Hydroxysafflor yellow A(HSYA)is the prime active element of safflower yellow pigment and the most effective water-soluble ingredient in the pharmacological efficacy of medicinal safflower.Hydroxy safflower yellow pigment A has been recognized for improving blood rheology and coagulation.Previous studies have shown that HSYA plays an important role in scavenging oxidative stress and inflammation,as well as inhibiting apoptosis in liver,brain,lung disease and cardiomyopathies.JNK signaling pathway is an important pathway involved in the mechanism of oxidative stress in diabetes.In islet β cells in diabetes,oxidative stress can lead to JNK activation,which leads to a decrease in insulin gene expression levels,disrupts insulin synthesis,and leads to apoptosis.However,there are few studies on the effects and mechanisms of HSYA on islet cells.In this experiment,we used a chronic high glucose-induced glucose toxicity model to explore the relationship between HSYA,oxidative stress,apoptosis,and islet function,and finally examined the JNK/c-jun regulatory pathway to reveal possible underlying mechanisms.Part I:Hydroxysafflor yellow A improves apoptosis of pancreatic βcells under high glucose toxicity by reducing oxidative stressObj ectiveThe section intends to establish an in vitro model of islet cell hyperglycemia.First,we verify the effect of HSYA on islet cell apoptosis under high glucose conditions,then,to observe whether this improvement is related to anti-oxidative stress,which provide a theoretical basis for its clinical application and experimental basis.MethodINS-1 cells were divided into the following groups:control group,high glucose group,HSYA concentration group for 72h.The MTT assay was used to estimate the changes of cell viability.The PDX-1 mRNA and insulin mRNA were detected by RT-PCR to reflect the insulin transcription.Cleaved-caspase3 and cleaved-parp were detected by western blotting to reflect the apoptosis.Randomly grouping after determining the appropriate concentration.Group 1:Treatment with 11.1mM glucose as control.Group 2:Treatment with 33.3 mM glucose.Group 3:Cells were pretreated with NAC at a dose of 5mM for 2h,and then treated with high glucose for 72 h.Group 4:Cells were treated by high glucose(33.3 mM)and HSYA(800uM)for 72 h.Western blotting was used to detect cleaved-caspase3 and cleaved-parp to reflect apoptosis.RT-PCR was used to detect the expression of CAT mRNA,SOD mRNA and GSH-px mRNA and to detect the content of CAT,GSH-px and MDA in cells.Microscopic observation of intracellular ROS production to reflect oxidative stress;RT-PCR detection of PDX-1 and insulin to reflect insulin transcription.Result1.Selection of HSYA concentrationCompared with the control group,the survival rate of INS-1 cells stimulated by high glucose for 72 h was significantly decreased(P<0.05)shown by MTT.HSYA protected cells from damage caused by high glucose in a concentration-dependent manner.RT-PCR showed that the PDX-1 mRNA and insulin mRNA levels were decreased under high glucose,and the mRNA was expressed gradually increased after deal with HSYA.Western blotting showed that the difference was statistically significant at the concentration of 800uM.We obtained 800 uM concentration of HSYA as a follow-up study.2.Effect of HSYA on apoptosis of INS-1 cellsCompared with the control group,the expression of cleaved-caspase3 and cleaved-parp was increased in the high-glycemic group;the expression of apoptotic protein was decreased after pre-administration of NAC;the expression of apoptoticprotein was decreased after HSYA administration.3.Effect of HSYA on oxidative stress index of INS-1 cells3.1 ROS contentThe high glucose group had higher fluorescence intensity than the control group.The ROS fluorescence intensity decreased after treatment with NAC antioxidant,and the intensity decreased after HSYA treatment.3.2 CAT,SOD,GSH-px,MDA expressionCompared with the control group,high glucose group showed a significant increase in MDA,and decreased in CAT,SOD,GSH-px both in mRNA and intracellular contents.After HSYA intervention,this phenomenon could be reversed.Treated with NAC,the group results are similar.4.Effect of HSYA on islet function of INS-1 cellsCompared with the control group,the PDX-1 mRNA and insulin mRNA levels were decreased in the high glucose group.The PDX-1 mRNA and insulin mRNA levels were increased after NAC administration.The PDX-1 mRNA and insulin mRNA levels were increased after HSYA administration.Conclusion1.HSYA can improve the apoptotic state of pancreatic β cells in high glucose state.2.Reducing oxidative stress may be one of the mechanisms by which HSYA improves the apoptotic state of pancreatic β cells.Part Ⅱ:JNK/c-jun pathway is involved in Hydroxysafflor yellow A to alleviate oxidative stress and the glucose toxicity of pancreatic β cellsObj ectiveIn this part,we intend to establish an in vitro model of high glucose toxicity of islet cells.Firstly,we verify the effect of HSYA on islet cells under high glucose conditions,and then observe whether this improvement is related to anti-oxidative stress.Finally,we investigate possible pathway of the anti-oxidative stress of HSYA on islet cells which provides a scientific basis for its clinical application.MethodINS-1 cells were divided into the following groups:(a)normal control group,(b)high glucose group,(c)high glucose + HSYA group,(d)high glucose +anisomycin group(JNK activator),(e)high glucose + HSYA+ anisomycin group for 72h.Cleaved-Caspase3 and cleaved-parp were used to reflect the apoptosis by western blotting.RT-PCR was used to detect the expression of CAT mRNA,SOD mRNA and GSH-px mRNA,and the content of CAT,GSH-px and MDA in cells were detected.Semi-quantitative detection of ROS levels were detected by fluorescence microscopy to reflect the oxidative stress;PDX-1 and insulin were detected by RT-PCR to reflect insulin transcription and Elisa was used to detect GSIS to reflect islet function.Result1.The effect of HSYA on apoptosis of INS-1 cellsThe expression of apoptotic protein was increased in the high glucose group and was decreased after HSYA administration.The expression of apoptotic protein was increased in the high glucose+anisomycin group compared with the high glucose group;the high glucose+HSYA+anisomycin group was higher than the high glucose+HSYA group.2.Effect of HSYA on oxidative stress index of INS-1 cells2.1 ROS contentThe high glucose group showed a significant increase in ROS levels compared with the control group,and the intensity after HSYA treatment decreased significantly,but the ROS increased significantly when HSYA was administered and anisomycin was administered in advance.2.2 CAT,SOD,GSH-px,MDA expressionCompared with the control group,high glucose group showed a significant increase in MDA,and decreased in CAT,SOD,GSH-px both in mRNA and intracellular contents.However,after HSYA intervention,this phenomenon can be reversed,but when anisomycin was administered in advance while HSYA was administered,the effect of anti-oxidative stress was significantly attenuated.3.Effect of HSYA on islet function of INS-1 cellsCompared with the control group,PDX-1 mRNA and insulin mRNA levels were decreased in the high glucose group.PDX-1 mRNA and insulin mRNA levels were increased after HSYA administration.However.PDX-1 mRNA and insulin mRNA levels were reduced when HSYA was administered with anisomycin in advance.However,although the level of mRNA was statistically significant,the difference in GSIS results was not statistically significant.Conclusion1.HSYA can improve the apoptotic state of pancreatic β cells in high glucose state.2.Reducing oxidative stress may be one of the mechanisms by which HSYA improves the apoptotic state of pancreatic β cells.3.The JNK/c-jun pathway may be involved in the oxidative stress of HSYA in pancreatic β cells.