The Role Of Klotho Protein In Hippocampal Cells Damage Induced By Isoflurane Exacerbated By High Glucose

Background and objective:In recent years,the effects of inhalation general anesthesia on postoperative Delayed neurocognitive recovery(PDNR)in diabetic patients have been drawing broad attention.As a kind of inhalation anesthetics which is commonly used,isoflurane can cause neurotoxic reactions to hippocampal cells of mice,such as oxidative stress and apoptosis,which end up affecting cognitive function.While the specific molecular mechanism involved is unknown.klotho protein is considered as an anti-aging protein.Its major function is regulating metabolism of calcium and phosphorus in renal tubular epithelial cells.And beyond that,it might also paly an important part in neuroprotection.Studies have shown that high glucose can reduce the expression of klotho protein in peripheral cells,but its effect on hippocampal neurons has not been reported.In this study,we are primarily exploring the role of klotho protein in hippocampal cells damage induced by isoflurane aggravated by high glucose,so as to provide a new diagnosis and treatment target for the prevention and treatment of PDNR complications in patients with diabetes after inhalation general anesthesia.Methods:Mouse hippocampal neuron(HT22)was cultured in vitro.In general,the cells were divided into control group(CON group),high glucose group(HG72h group),isoflurane group(ISO group),isoflurane group,high glucose plus+isoflurane group(HG+ISO group),over-expression klotho+ high glucose group(HG/klotho group),over-expression klotho+isoflurane group(ISO/klotho group),over-expression klotho+high glucose+isoflurane group(HG+ISO/klotho group).CCK-8 method was used to detect cell viability,ROS staining was used to detect the level of reactive oxygen species,JC-1 staining was used to detect mitochondrial membrane potential changes,Hoechst 33342 staining was used to detect the degree of early apoptosis,and Western blot was used to detect klotho protein expression level and Bcl-2/Bax protein ratio.Results:(1)After cultured with high glucose,the level of klotho protein expression and Bcl-2/Bax protein ratio of HT22 cells were decreased,the level of ROS was increased,mitochondrial membrane potential was decreased,and the cell nucleus showed shrinkage and was highlighted by Hoechst 33342 staining.(2)After cultured with isoflurane,the level of klotho protein expression of HT22 cells was increased,and the trend of results of Bcl-2/Bax protein ratio,ROS,JC-1 and Hoechst 33342 staining was consistent with which of HG group.(3)Compared with the ISO group,the level of klotho protein expression was decreased in HG+ISO group,and the results of ROS,JC-1 and Hoechst 33342 staining were all aggravated.However,there was no significant difference in Bcl-2/Bax protein ratio between the two groups.(4)After over-expression of klotho protein,the adverse results of each groups were reduced.Conclusion:(1)High glucose can decrease the expression of klotho protein in HT22 cells,and induce the oxidative stress and apoptosis.(2)The pretreatment of high glucose can aggravate the oxidative damage and apoptosis of HT22 cells induced by isoflurane;The overexpression of klotho protein can reduce the damage of HT22 cells induced by high glucose and isoflurane.