PGD2-DPs Pathway Is Involved In The T2DM Brain Injury By Regulating Autophagy

Part I The change of PGD2-DP_S pathway and autophagy level in the T2DM brain injuryAim:To observe the changes of PGD2-DPs pathway and autophagy level in cerebral cortex and hippocampus of rats with diabetic brain injury.COX2 inhibitor was used to reduce the content of PGD2,and its effect on the brain injury of diabetic rats was observed.It was planned to preliminarily observe whether the changes of PGD2-DPs pathway and autophagy level were correlated with the mechanism of diabetic brain injury.Method:1.60 male SD rats（80-100g）were randomly divided into the normal group（n=10）and model group（n=50）.Rats in the model group were fed a high-fat and high-sugar diet for one month,and STZ（40mg/kg）was intraperitoneally injected once to establish diabetes model.After successful modeling,30 diabetic rats were randomly selected to divide into three groups:model group（n=10）,model+low-dose meloxicam group(1mg·kg^-1)（n=10）and model+high-dose meloxicam group(3 mg·kg^-1)（n=10）.The normal and model group rats were administrated with 0.5%CMC-Na by intragastric,and the model+meloxicam group rats was given meloxicam by intragastric for the lasting 8 weeks.All groups were fed with normal diet for 8 weeks.2.The blood glucose was measured by blood glucose meter.The levels of TG,LDL,and TC in rat plasma were measured by kit.The learning and memory function were evaluated by Morris water maze.HE staining was used to detect the morphologic change of neurons.The insulin content in plasma and PGD2 content in rat cortex and hippocampus were detected by ELISA.Western blotting was used to detect the expression of COX2,DP1,DP2,p62 and LC3BII in rat cortex and hippocampus.Result:1.When the administration was stopped,there were 9 rats survived in model group and COX2 inhibitor group.Compared with the normal group,the blood glucose levels were significantly increased in the model group（P<0.01）;the levels of TG and TC were significantly increased（P<0.01）,and there was no significant change in the level of LDL in the model group;the morris water maze test showed that the escape latency significantly prolonged and the crossing times significantly decreased in the model group（P<0.01）;the cortex and hippocampus neurons were showed significantly karyopyknosis in the model group;the plasma insulin content was significantly decreased in the model group（P<0.01）;the PGD2 content was significantly increased in the model rat hippocampus and cortex（P<0.01）;the expressions of COX2（P<0.01）,DP2（P<0.05）and p62（P<0.01）were significantly increased,whereas the expression of DP1（P<0.01）and LC3BII（P<0.01）was significantly decreased in the model rat hippocampus and cortex.2.Compare with the model group,the COX2 inhibitor has no effect on the blood glucose,TG,TC,LDL,and plasma insulin in moedel+COX2 inhibitor group;the COX2 inhibitor significantly decreased the escape latency and increased the crossing times（P<0.01）in moedel+COX2 inhibitor group;the COX2 inhibitor significantly improved neurons karyopyknosis in moedel+COX2 inhibitor group;the COX2inhibitor significantly decreased the content of PGD2（P<0.01）and the expression of COX2（P<0.01）and p62（P<0.01）,while significantly increased the expression of LC3BII（P<0.01）in moedel+COX2 inhibitor group.Conclusion:The results suggest that the PGD2-DPs pathway is abnormal and autophagy level is decreased in the cortex and hippocampus of rats with diabetic brain injury.The inhibition of COX2 reduces the content of PGD2,thereby increasing autophagy level,which may have a protective effect on brain injury in diabetic rats.Part II The change of PGD2-DP_S pathway and autophagy level in HT22 cells treated with high glucoseAim:To observe the changes of PGD2-DPs pathway and autophagy level in high glucose treatment of HT22 cells.The COX2 inhibitor was used to reduce the content of PGD2,and to observe the correlation between the changes of PGD2-DPs pathway and autophagy level and the mechanism of high glucose-induced HT22 cell injury in vitro.Method:1.High glucose-damaged HT22 cells model was established:high glucose medium（75mM）was applied to HT22 cells for 36h（HG group）.The HG group was treated with a COX2 inhibitor（meloxicam）,an autophagy inhibitor（wortmannin）and an autophagy agonist（rapamycin）,respectively.2.The content of PGD2 in cells was detected by ELISA.The number of autophagosomes in HT22 cells was detected by transmission electron microscopy.The HT22 cells survival rate was detected by MTT.The HT22 cells death rate was detected by LDH.The HT22 cells apoptosis and necrosis rate were detected by flow cytometry.The expresson of COX2,DP1,DP2,p62 and LC3BII were detected by western blotting.Result:1.Compare with the control group,the number of autophagosomes was significantly increased in HG group;the HT22 cells survival rate（P<0.01）and the HT22 cell numbers were significantly decrased in HG group;the HT22 cells LDH leakage rate（P<0.01）,apoptosis and necrosis rate were significantly increased in HG group;the PGD2 content was significantly incrased（P<0.01）in HG group;the expressions of COX2（P<0.01）,DP1（P<0.01）,DP2（P<0.05）and p62（P<0.05）were significantly increased,whereas the expression of LC3BII（P<0.01）was significantly decreased in HG group.2.Compare with the HG group,the wortmannin significantly decreased the HT22cells survival rate（P<0.05）in HG+wortmannin group;the rapamycin significantly increased the HT22 cells survival rate（P<0.05）in HG+rapamycin group;the COX2inhibitor significantly increased the HT22 cells survival rate（P<0.05）and the HT22cells numbers in moedel+COX2 inhibitor group;the COX2 inhibitor significantly decreased the content of PGD2（P<0.01）in moedel+COX2 inhibitor group;the COX2inhibitor significantly decreased the HT22 cells LDH leakage rate（P<0.05）,apoptosis and necrosis rate in moedel+COX2 inhibitor group;the COX2 inhibitor significantly decreased the expression of COX2（P<0.05）and p62（P<0.05）,while significantly increased the expression of LC3BII（P<0.05）in HG+COX2 inhibitor group.Conclusion:The results suggest that the PGD2-DPs pathway is abnormal and autophagy level is decreased in high glucose treatment of HT22 cells.COX2 inhibitors have protective effects on high glucose-induced HT22 cells,which may be involved in decreasing PGD2 to increased autophagy level.Part III Effect of PGD2-DP2 regulating autophagy on high glucose-induced HT22 cells injuryAim:To observe the effect of DP2 intervention on high glucose-induced HT22 cell injury,and to determine whether its mechanism affects autophagy regulation through PI3K/AKT/mTOR pathway.Method:1.High glucose medium（75mM）was administered to HT22 cells for 36 h,the DP2 agonist and inhibitor were administered,respectively.The autophagy promoter and inhibitor were administered to DP2 agonists and inhibitors,respectively.The experimental were divided into six groups:normal group,HG group,HG+DP2 agonist group,HG+DP2 agonist+rapamycin group,HG+DP2 inhibitor group,HG+DP2inhibitor+wortmannin group.2.The number and morphology of HT22 cells were observed by microscopy.The HT22 cells survival rate was detected by MTT.The HT22 cells death rate was detected by LDH.The HT22 cells apoptosis and necrosis rate were detected by flow cytometry.The expression of PI3K,AKT,p-AKT（s473）,mTOR,p-mTOR（s2448）,p62 and LC3BII were detected by western blotting.Results:1.Compare with the HG group,the HT22 cells survival rate（P<0.05）was significantly decreased and the HT22 cells LDH leakage rate（P<0.01）was significantly increased in HG+DP2 agonist group;the HT22 cells survival rate（P<0.05）was significantly increased and the HT22 cells LDH leakage rate（P<0.05）was significantly decreased in HG+DP2 inhibitor group.Compared with the HG+DP2 agonist group,the HT22 cells survival rate（P<0.05）was significantly increased and the HT22 cells LDH leakage rate（P<0.01）was significantly decreased in HG+DP2 agonist+rapamycin group.Compared with the HG+DP2 inhibitor group,the HT22 cells survival rate（P<0.05）was significantly decreased and the HT22 cells LDH leakage rate（P<0.01）was significantly increased in HG+DP2 inhibitor+wortmannin group.2.The DP2 agonist significantly decreased the number of HT22 cells in HG group.The DP2 inhibitor significantly increased the number of HT22 cells in HG group.Compared with the HG+DP2 agonist group,rapamycin significantly increased the number of HT22 cells in HG+DP2 agonist+rapamycin group.Compared with the HG+DP2 inhibitor group,wortmannin significantly decreased the number of HT22cells in HG+DP2 inhibitor+wortmannin group.3.Compare with the HG group,DP2 agonist significantly increased the HT22cells necrosis and apoptosis rate in HG+DP2 agonist group;the DP2 inhibitor significantly decreased the HT22 cells necrosis and apoptosis rate in HG+DP2inhibitor group.Compared with the HG+DP2 agonist group,rapamycin significantly decreased HT22 cells necrosis and apoptosis rate in HG+DP2 agonist+rapamycin group.Compared with the HG+DP2 inhibitor group,wortmannin significantly decreased the HT22 cells necrosis and apoptosis rate in HG+DP2 inhibitor+wortmannin group.4.Compared with the control group,the expression of PI3K（P<0.05）,AKT（P<0.01）,p-AKT（s473）（P<0.01）,mTOR（P<0.05）,p-mTOR（s2448）（P<0.05）and p62（P<0.01）were significanly increased,while the expression of LC3BII（P<0.05）was significanly decreased in HG group.Compared with the HG group,the DP2agonist significantly increased the expression of PI3K（P<0.05）,AKT（P<0.05）,p-AKT（s473）（P<0.05）,mTOR（P<0.05）,p-mTOR（s2448）（P<0.05）and p62（P<0.01）,and decreased the expression of LC3BII（P<0.05）in HG+DP2 agonist group;the DP2inhibitor significantly decreased the expresssion of PI3K（P<0.05）,AKT（P<0.05）,p-AKT（s473）（P<0.05）,mTOR（P<0.05）,p-mTOR（s2448）（P<0.05）and p62（P<0.05）,while decreased the expression of LC3BII（P<0.05）in HG+DP2 inhibitor group.Compared with the HG+DP2 agonist group,the rapamycin significantly decreased the expression of mTOR（P<0.05）,p-mTOR（s2448）（P<0.01）and p62（P<0.01）,and significantly increased the expression of LC3BII（P<0.05）,while the expression of PI3K,AKT and p-AKT（s473）had no significant changes in HG+DP2agonist+rapamycin group.Compared with the HG+DP2 inhibitor group,wortmannin significantly increased the expression of p62（P<0.01）,and decreased the expression of LC3BII（P<0.05）,while the expression of PI3K,AKT,p-AKT（s473）,mTOR and p-mTOR（s2448）had no significant changes in HG+DP2 inhibitor+wortmannin group.Conclusion:In the HG group,the DP2 could activate the PI3K/AKT/mTOR pathway to inhibit autophagy to promote the damage of HG.Part IV Effect and autophagy regulation mechanism of DP1 in high glucose-damaged HT22 cellsAim:To observe the effect of DP1 intervention on high glucose-induced HT22 cell injury,and to preliminarily determine whether the mechanism is through the PKA/mTOR pathway and then regulate autophagy.Method:1.The effect of DP1 intervention on high glucose-induced HT22 cell injury,autophagy level and cAMP/PKA-C/mTOR pathway:（1）High glucose medium（75 M）was administered to HT22 cells for 36 h,the DP1 agonist and inhibitor were administered,respectively.The autophagy promoter was administered to DP1 inhibitors.The experimental were divided into five groups:normal group,HG group,HG+DP1 agonist group,HG+DP1 inhibitor group,and HG+DP2 inhibitor+rapamycin group（only for MTT,LDH and flow cytometry experiments）.（2）The number and morphology of cells were observed by microscopy.The HT22 cells survival rate was detected by MTT.The HT22 cells cell death rate was detected by LDH.The HT22 cells apoptosis and necrosis rate were detected by flow cytometry.The content of cAMP was detected by ELISA.The expression of PKA-C（α/β）,mTOR,p-mTOR（s2448）,p62 and LC3BII were detected by western blotting.2.To observe whether DP1 regulates mTOR expression through PKA-C:（1）The experiment was divided into normal group,normal+no-loading lentivirus group,HG group,HG+no-loading lentivirus group,HG+PKA-C（α/β）shRNA（respectively silencedαandβ）group,HG+DP1 agonist,HG+PKA-C（α/β）shRNA（respectively silencedαandβ）+DP1 agonist group.The effect of no-loading lentivirus on the survival of normal cells was detected by flow cytometry.The expression of PKA-C（α/β）,mTOR and p-mTOR（s2448）protein expression were detected by western blotting.（2）To observe interaction between PKA-C and mTOR:the Co-IP experiments was used to detect whether PKA-C（α/β）and mTOR have direct binding.Result:1.The effect of DP1 intervention on high glucose-induced HT22 cell injury,autophagy level and cAMP/PKA-C/mTOR pathway:（1）Compare with the HG group,the HT22 cells survival rate（P<0.05）was significantly increased and the HT22 cells LDH leakage rate（P<0.05）was significantly decreased in HG+DP1 agonist group;the HT22 cells survival rate（P<0.05）was significantly decreased and the HT22 cells LDH leakage rate（P<0.05）was significantly increased in HG+DP1 inhibitor group.Compared with the DP1 inhibitor group,the HT22 cells survival rate was significantly increased（P<0.05）and the leakage rate of LDH was significantly decreased（P<0.05）in HG+DP1 inhibitor+rapamycin group.（2）The DP1 agonist significantly increased the number of cells in HG group.The DP1 inhibitor significantly decreased the number of cells in HG group.Compared with the DP1 inhibitor group,the number of cells was significantly increased in HG+DP1inhibitor+rapamycin group.（3）Compared with the control group,the content of cAMP was significantly increased in the HG group（P<0.05）.Compared with the HG group,the the content of cAMP was significantly increased in HG+DP1 agonist group（P<0.05）,and the content of cAMP was significantly decreased in HG+DP1 agonist group（P<0.01）.Compared with the HG group,DP1 agonist significantly decreased the rate of necrosis and apoptosis in HG+DP1 agonist group;the DP1 inhibitor significantly increased the rate of necrosis and apoptosis in HG+DP1 inhibitor group.Compared with the DP1inhibitor group,the rate of necrosis and apoptosis was significantly decreased in HG+DP1 inhibitor+rapamycin group.MTT,LDH,cell number and flow cytometry results showed that mTOR may be the DP1 downstream pathway protein.（4）Compared with the control group,the expression of PKA-C（α/β）（P<0.05）,mTOR（P<0.05）,p-mTOR（s2448）（P<0.05）and p62（P<0.01）were significantly increased,while the expression of LC3BII（P<0.01）were significantly decreased in HG group.Compared with the HG group,the expression of PKA-C（α/β）（P<0.05）and LC3BII（P<0.05）were significantly increased,the expression of mTOR（P<0.05）,p-mTOR（s2448）（P<0.05）and p62（P<0.05）were significantly decreased in HG+DP1agonist group;the expression of PKA-C（α/β）（P<0.05）and LC3BII（P<0.05）were significantly decreased,while expression of mTOR（P<0.05）,p-mTOR（s2448）（P<0.05）and p62（P<0.05）in significantly increased in HG+DP1 inhibitor group.2.DP1 affects mTOR expression through PKA-C:（1）Compared with the normal group,the expression of PKA-C（α/β）（P<0.01）,mTOR（P<0.01）and p-mTOR（s2448）（P<0.01）was significantly increased in HG group.Compared with HG group,the expression of PKA-C（α/β）（P<0.01）was significantly decreased,while the expression of mTOR（P<0.01）and p-mTOR（s2448）（P<0.01）were significantly increased in HG+PKA-C（α）shRNA group;the expression of PKA-C（α/β）（P<0.01）was significantly decreased,while the expression of mTOR（P<0.01）and p-mTOR（s2448）（P<0.01）were significantly increased in HG+PKA-C（β）shRNA group.（2）Compared with HG group,the expression of PKA-C（α/β）（P<0.05）was significantlyincreased,whiletheexpressionofmTOR（P<0.05）and p-mTOR（s2448）（P<0.05）were significantly decreased in HG+DP1 agonist group.Compared with the HG+DP1 agonist group,the expression of PKA-C（α/β）（P<0.05）was significantly decreased,while the expression of mTOR（P<0.05）and p-mTOR（s2448）（P<0.05）were significantly increased in HG+DP1 agonist+PKA-C（α）shRNA group;the expression of PKA-C（α/β）（P<0.05）was significantly decreased,while the expression of mTOR（P<0.05）and p-mTOR（s2448）（P<0.05）were significantly increased in HG+DP1 agonist+PKA-C（β）shRNA group.（3）Co-IP experiments prove that the catalytic subunit of PKA has direct binding with mTOR.Conclusion:（1）mTOR may be a downstream pathway protein of DP1;the catalytic subunit of PKA has direct binding to mTOR.（2）Both the catalytic subunitsαandβof PKA are involved in the inhibition of mTOR expression.（3）DP1 receptor promotes autophagy by activating PKA/mTOR pathway to improve high glucose-induced HT22 cell injury.