The Mechanism Of Diosgenin Based On Mitochondrial Autophagy To Improve Testicular Injury Caused By Lipid Metabolism Disorder In Type 2 Diabetes Mellitus

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The mechanism of diosgenin based on mitochondrial autophagy to improve testicular injury in type 2 diabetes mellitus ratsObjectiveTo explore whether diosgenin can improve testicular injury by affecting mitochondrial autophagy in testicular tissue of type 2 diabetes mellitus rats.MethodA rat model of testicular injury caused by dyslipidemia in type 2 diabetes mellitus was induced by high fat diet and streptozotocin injection,the experimental rats were randomly divided into model group,low-dose diosgenin group,medium dose diosgenin group,high-dose diosgenin group and metformin positive control group,and normal rats were set as normal control group.After 12 weeks of continuous drug intervention,the following related experimental indicators were detected and analyzed.Triglycerides(TG)and fasting blood glucose were measured by biochemical methods.Enzyme linked immunosorbent assay(ELISA)was used to measure the levels of serum insulin(INS)and testosterone.Hematoxylin eosin staining(HE)was used for pathological examination under light microscope and the microstructure was examined under electron microscope.The level of reactive oxygen species(ROS)in testis was measured by DHE fluorescent probe.JC-1fluorescent probe was used to detect the level of mitochondrial membrane potential(??m)in testicular cells.The opening of mitochondrial permeability transition pore(MPTP)in testicular cells was measured by Calcein-AM fluorescent probe.The content of cytochrome c(Cyt c)in testis was measured by ELISA.Quantitative reverse transcription PCR(RT-q PCR)was used to detect the expression of messenger RNA(m RNA)of steroidogenic acute regulatory proteins(St AR)in testis.Western blot(WB)was used to detect the expression of St AR protein,insulin signaling pathway related proteins IRS-1,p-IRS-1(Ser307),Akt,p-Akt(ser473),JNK,p-JNK(thr183/tyr185)and mitochondrial autophagy related proteins PINK1,Parkin,LC3-?,LC3-? and p62.ResultsCompared with the normal control group,the serum TG level of the model group was abnormally increased,the serum testosterone level was abnormally low,the testicular weight of the model group significantly reduced,the HE staining morphological examination showed that the testicular tissue cell structure pathological changes were obvious,the testicular tissue St AR m RNA and St AR protein expression levels significantly decreased.Compared with the model group,the serum TG level of diosgenin group and metformin positive control group significantly decreased,the serum testosterone level significantly increased,the testicular weight of diosgenin group and metformin positive control group significantly increased,the cell structure and pathology of testicular tissue significantly reduced,and the expression levels of St AR m RNA and St AR protein in testicular tissue significantly decreased.Compared with the normal control group,the level of ROS in the testicular tissue of the model group increased abnormally,MPTP abnormally opened,the level of ??m decreased significantly,and Cyt c in the cytoplasm of testicular tissue cells increased significantly.Compared with the model group,the level of ROS in the testicular tissue of diosgenin group and metformin positive control group decreased abnormally,the opening degree of MPTP decreased significantly,the level of ??m increased significantly,and Cyt c in the cytoplasm of testicular tissue cells decreased significantly.Compared with the normal control group,JNK(thr183/tyr185)phosphorylation level and IRS-1(Ser307)phosphorylation level of testicular tissue in the model group significantly increased,while Akt(ser473)phosphorylation level significantly decreased;Compared with the model group,JNK(thr183/tyr185)phosphorylation level and IRS-1(Ser307)phosphorylation level of testicular tissue in diosgenin group and metformin positive control group significantly reduced,while Akt(ser473)phosphorylation level significantly increased.The ultrastructure showed that the organelles in Leydig cells of normal control group were intact and clear,and no mitochondrial autophagosome was found.In the model group,the morphology of organelles in Leydig cells was not clear,and mitochondrial autophagosome deposits could be seen.In diosgenin group and metformin positive control group,the morphology of organelles in Leydig cells were complete and clear,and there were also mitochondrial autophagosome deposits.The expression levels of mitochondrial autophagy related proteins PINK1,Parkin,LC3-?/LC3-? and p62 of testicular tissue in normal control group were very low.Compared with the normal control group,the expression levels of PINK1,Parkin,LC3-?/LC3-? and p62 in the model group significantly increased,especially p62.Compared with the model group,the expression levels of mitochondrial autophagy related proteins PINK1,Parkin,LC3-?/LC3-? in diosgenin group and metformin positive control group significantly increased,while the expression levels of p62 significantly decreased.ConclusionThe dysfunction of PINK1-Parkin signaling pathway dependent mitochondrial autophagy induced by ROS may be an important pathophysiological mechanism of testicular injury caused by lipid metabolism disorder in type 2 diabetes mellitus rats.Both diosgenin and metformin have therapeutic effects on testicular injury caused by lipid metabolism disorder in type 2 diabetes mellitus rats.The mechanisms of diosgenin are related to the facts that diosgenin can activate PINK1-Parkin signaling pathway dependent mitochondrial autophagy,improve the dysfunction of PINK1-Parkin signaling pathway dependent mitochondrial autophagy,clear the ROS in testicular tissue of type 2 diabetes mellitus rats with testicular injury caused by dyslipidemia.Section ? The mechanism of diosgenin based on mitochondrial autophagy to improve palmitic acid-induced Leydig cell injuryObjectiveTo explore whether diosgenin can improve palmitic acid-induced Leydig cell injury by affecting mitochondrial autophagy.MethodThe damage model of TM3 cells induced by palmitic acid(PA)was established.In the part of diosgenin mechanism study,we also prepared recombinant lentivirus targeting PINK1 si RNA to transfect TM3 cells,then PA stimulation and drug intervention were carried out.In the part of diosgenin pharmacodynamic study,the experimental groups were divided into normal control group,model group,1?M diosgenin treatment group,3?M diosgenin treatment group,10?M diosgenin treatment group and 10 ?M metformin positive control group.The pharmacodynamic indexes were as follows: lipid in TM3 cells was observed by oil red O staining,TG content was measured by biochemical method,glucose consumption level was detected by fluorescence method,and testosterone secretion level was detected by ELISA.In the part of diosgenin mechanism study,the experimental groups were divided into normal control group,model group,10?M diosgenin treatment group,(PA+empty lentivirus)group,(PA+si PINK1 lentivirus)group,(PA+si PINK1 lentivirus+10?M diosgenin)group,(PA+10 ?M diosgenin+ROS scavenger N-acetylcysteine)group,(PA+si PINK1 lentivirus+10 ?M diosgenin+ROS scavenger N-acetylcysteine)group.The mechanism indexes were as follows:The level of ROS in TM3 cells was measured by DHE fluorescent probe,the level of ??m was measured by JC-1 fluorescent probe,the opening degree of MPTP was measured by Calcein-AM fluorescent probe,and the content of adenosine triphosphate(ATP)was measured by luciferase catalytic method,RT-q PCR was used to detect the expression of St AR m RNA,WB was used to detect the expression of St AR protein,insulin signaling pathway related proteins IRS-1,p-IRS-1(Ser307),Akt,p-Akt(ser473),JNK,p-JNK(thr183/tyr185)and mitochondrial autophagy related proteins PINK1,Parkin,LC3-?,LC3-? and p62.ResultsIn this part of diosgenin pharmacodynamic experiment,compared with the normal control group,the TG content of TM3 cells in the model group abnormally increased,the lipid deposition was obvious,the glucose consumption significantly decreased,and the testosterone secretion level abnormally decreased;compared with the model group,the TG content of TM3 cells in the Diosgenin treatment group and the metformin positive control group significantly decreased,the lipid deposition was significantly reduced,the glucose consumption significantly decreased,and the level of testosterone secretion increased significantly.In this part of diosgenin mechanism experiment,compared with the normal control group,the level of ROS in TM3 cells of model group and(PA+empty lentivirus)group increased abnormally,and MPTP abnormally opened in both groups.The level of ??m decreased significantly,the content of ATP decreased significantly,and the expression levels of PINK1,Parkin,LC3-?/LC3-? and p62 proteins increased significantly,especially p62 protein.Therefore,the phosphorylation levels of JNK(thr183/tyr185)and IRS-1(Ser307)abnormally increased,Akt(ser473)phosphorylation levels abnormally decreased,and the expression levels of St AR m RNA and St AR protein significantly decreased.Compared with the model group,the level of ROS in TM3 cells of(PA+si PINK1 lentivirus)group abnormally increased,the abnormal opening degree of MPTP increased,the level of ??m significantly decreased,and the content of ATP significantly reduced.The expression level of PINK1 significantly decreased.However,there was no significant difference in the expression of Parkin,LC3-?/LC3-? and p62.Therefore,the phosphorylation levels of JNK(thr183/tyr185)and IRS-1(Ser307)abnormally increased,while the phosphorylation levels of Akt(ser473)were not significantly different.The expression levels of St AR m RNA significantly decreased,and the expression levels of St AR protein were not significantly different.In this part of diosgenin mechanism experiment,compared with the model group,the TM3 cells of 10?M diosgenin treatment group and(PA+10 ?M diosgenin+ROS scavenger N-acetylcysteine)group showed significant decrease in ROS level and MPTP opening degree,and significant increase in ??m and ATP content,the expression levels of PINK1,parkin and LC3-?/LC3-? significantly increased,but the expression levels of p62 significantly decreased.Therefore,the phosphorylation level of JNK(thr183/tyr185)and IRS-1(Ser307)significantly reduced,Akt(ser473)phosphorylation level significantly increased,while the expression level of St AR m RNA and St AR protein significantly increased.In this part of diosgenin mechanism experiment,compared with the model group,the level of ROS in TM3 cells of(PA+si PINK1 lentivirus+10 ?M diosgenin)group and(PA+si PINK1 lentivirus+10 ?M diosgenin+ROS scavenger N-acetylcysteine)group abnormally increased,the abnormal opening degree of MPTP increased,the average level of ??m significantly decreased,the content of ATP significantly decreased,the expression level of mitochondrial autophagy related protein PINK1 significantly decreased,the expression levels of mitochondrial autophagy related protein Parkin and LC3-?/LC3-? were not significantly different,but the expression level of p62 protein significantly decreased.Therefore,the phosphorylation levels of JNK(thr183/tyr185),IRS-1(Ser307)and Akt(ser473)were not significantly different,while the expression levels of St AR m RNA and St AR protein decreased.There was no significant difference in St AR protein expression.ConclusionThe dysfunction of PINK1-Parkin signaling pathway dependent mitochondrial autophagy induced by ROS may be an important pathophysiological mechanism of PA induced TM3 cell injury.Diosgenin has a significant therapeutic effect on PA induced TM3 cell injury.The mechanisms of diosgenin are related to the facts that diosgenin can activate PINK1-Parkin signaling pathway dependent mitochondrial autophagy,improve the dysfunction of PINK1-Parkin signaling pathway dependent mitochondrial autophagy,clear the ROS in TM3 cell with injury caused by PA.