Effect And Mechanism Of Metformin Targeting AMPK/TFEB/LAMP1 Axis On Antioxidant Damage Of Granulosa Cells And Mouse Ovary

PurposePremature ovarian insufficiency(POI)is a complex reproductive endocrine disease,with a global incidence of about 1%,which seriously affects women's physiological and reproductive health.As there is no effective treatment method for POI widely applied in clinical practice,it is of great significance to explore specific targets for effective treatment of infertility and prevention of long-term complications in POI patients,and to develop new treatment strategies.Studies have found that metformin has potential anti-aging effects and reduces the risk of subsequent POI amongst patients with a history of polycystic ovarian syndrome(PCOS).To investigate whether metformin has a potential therapeutic effect on POI,we explored whether metformin can enhance the anti-oxidant damage function of granulosa cells and POI model mice ovary and its possible regulatory mechanism through in vitro cell experiment and in vivo animal model experiment,in order to provide new ideas and methods for the application of metformin and early prevention and treatment of POI.MethodsIn vitro cell experiment:(1)First,H₂O₂ was used to induce damage and CCK8 was used to detect cell viability.Then,a model of oxidative stress injury of granulosa cells was established.Next,we use CCK8 to detect the toxic effect of metformin on cells,and screen the best concentration of metformin to protect cells from oxidative damage.ROS in KGN cells was detected by DCFH-DA fluorescence probe,mitochondrial membrane potential was detected by JC-1 fluorescence probe,apoptosis was detected by Annexin V/PI double staining(flow cytometry)and TUNEL,superoxide dismutase(SOD)activity was detected by WST-1 method,lipid peroxidase(MDA)content was detected by TBA method,AMPK and P-AMPK,autophagy-related proteins LC3B and SQSTM1,and apoptosis-related proteins P53 and Bax were detected by Western blot.Autophagic flux was detected by Stub RFP-sens GFP-LC3 double fluorescent adenovirus infection to explore the protective effect of metformin on antioxidant damage of granulosa cells.The cells were treated with Compound C,an inhibitor of AMPK,and the expression of ROS,mitochondrial membrane potential,apoptosis,AMPK,P-AMPK,LC3B,SQSTM1,P53 and Bax were detected.It is suggested that metformin regulates autophagy homeostasis by activating AMPK and plays a protective role in cells.(2)The effects of metformin and Compound C on TFEB expression in oxidative-damaged granulosa cells were detected by western blot.The effects of metformin on the expression of TFEB,LC3B and SQSTM1 induced by H₂O₂ were detected after silencing AMPK.The effects of metformin on autophagic flux of oxidative-damaged cells were observed after silencing AMPK with Stub RFP-sens GFP-LC3 double fluorescent adenovirus.PRKAA2 overexpression plasmid was transfected into cells to observe the effect of AMPK on TFEB expression.Co-Immunoprecipitation(Co-IP)was used to detect the interaction between AMPK and TFEB.Immunofluorescence and western blot were used to detect the effect of metformin on the subcellular localization of oxidative-damaged granulosa cells.(3)Lyso-Tracker Red fluorescence probe was used to detect lysosomes and western blot was used to detect the expression of LAMP1.The effect of metformin on the number and function of lysosomes in oxidative damaged cells was observed.TFEB was silenced and LAMP1 gene expression was detected by q RT-PCR.TFEB was silenced and the effects of metformin on the expression of LAMP1,LC3B and SQSTM1 protein in oxidative damaged cells were detected.TFEB was silenced and immunofluorescence was used to detect the effect of metformin on lysosomes of oxidative damaged cells.TFEB overexpression plasmid was transfected into cells to observe the effect of TFEB on LAMP1 expression in oxidative damaged cells.AMPK was silenced,and the effect of metformin on LAMP1 expression in oxidative damaged cells was observed.The abundance of TFEB binding LAMP1 promoter region was detected by CHIP-PCR.POI animal model experiment:C57BL/6J female mice,7-8 weeks old,75 mice with normal estrous cycle were selected as the research subjects and divided into five groups.Normal control group,D-gal model group,metformin low-dose treatment group,metformin middle-dose treatment group,metformin high-dose treatment group,all treated for 42 days.Vaginal exfoliated cell smears were used to observe the changes of estrous cycle in mice.Serum sex hormones FSH,LH,E₂ and AMH were detected by ELISA.Serum ATP levels were detected.Ovarian morphology and follicle count were observed by hematoxylin-eosin(HE)staining.Ovarian granulosa cell apoptosis was detected by TUNEL method.SOD activity and MDA content in ovarian tissue were detected by WST-1 and TBA methods respectively.Western blot was used to detect AMPK,P-AMPK,LC3B,SQSTM1,TFEB,LAMP1,P53 and Bax protein expression levels in ovarian tissue of mice.ResultsIn vitro cell experiment:(1)The oxidative stress cell model was successfully established,and the best concentration of metformin to protect cells from oxidative damage was screened out.Metformin can reduce ROS,increase mitochondrial membrane potential,decrease apoptosis,increase SOD activity and decrease MDA level.Metformin can restore the blocking of autophagic flux,promote the expression of P-AMPK,and reduce the expression of autophagy-related proteins LC3B and SQSTM1 and apoptosis-related proteins P53 and Bax.The protective effects of metformin on ROS,mitochondrial membrane potential and apoptosis were reversed after treatment with Compound C,an AMPK inhibitor.At the same time,the expression of LC3B and SQSTM1 and apoptosis-related proteins P53 and Bax were reversed.(2)Metformin promoted TFEB expression in oxidative-damaged cells,and Compound C significantly inhibited the effect of metformin on TFEB expression.Silencing of PRKAA2 significantly inhibited the expression of TFEB,eliminated the enhancement of TFEB expression by metformin,and inhibited the effect of metformin on the expression of LC3B II/I and SQSTM1 in oxidative damaged cells.Silencing of PRKAA2 inhibited the recovery of autophagic flux by metformin.After overexpression of AMPK,the expression of TFEB increased,which reversed the expression of TFEB in oxidative damaged cells and showed similar regulatory effect to metformin.Co-IP detection proved the interaction between AMPK and TFEB.Immunofluorescence and western blot showed that metformin promoted the nuclear translocation of TFEB.(3)Metformin can reverse the lysosomal damage induced by H₂O₂,increase the number of lysosomes,promote the expression of LAMP1 in oxidative damaged cells,and improve the lysosomal function.Silencing of TFEB down-regulated the expression of LAMP1 m RNA and LAMP1 protein,and inhibited the enhancement of LAMP1expression and the down-regulation of LC3B II/I and SQSTM1 expression by metformin.Silencing of TFEB inhibited the protective effect of metformin on injured lysosomes.Overexpression of TFEB can increase the expression level of LAMP1 and reverse the down-regulation of LAMP1 induced by H₂O₂,which is similar to metformin.Silencing of AMPK caused the decrease of LAMP1 expression and inhibited the up-regulation of metformin on LAMP1.CHIP-PCR results showed that TFEB was bound to the 1823-2007bp region of LAMP1 gene promoter in two regions amplified by q PCR.Moreover,oxidative damage decreased the binding force between TFEB and LAMP1gene promoter region,while metformin pretreatment increased the binding force between TFEB and LAMP1 gene promoter region.POI animal model experiment:POI mouse model was successfully established with D-gal.The middle and high dose metformin treatment groups can improve the estrous cycle of mice.Compared with the model group,the low,medium and high dose metformin treatment groups can reduce serum FSH and increase AMH and E₂ levels in mice.The middle and high doses of metformin treatment group can improve the ovarian index of mice,improve the morphology of ovarian tissue,increase the number of antral follicles,decrease the number of atresia follicles,reduce the apoptosis of ovarian granulosa cells,increase the activity of SOD in ovarian tissue and decrease the level of MDA in POI mice.As for the metformin treatment group,compared with the model group,the low,middle and high dose treatment groups can increase the expression level of p-AMPK/AMPK,and the low,medium and high dose treatment groups can increase TFEB expression.The expression level of LAMP1 was increased in middle and high dose groups,and the expression of SQSTM1 protein was decreased in low,middle and high dose groups.Meanwhile,the expression level of apoptosis protein P53 and Bax was decreased in middle and high dose groups,which further suggested that middle and high dose metformin could improve the damage of ovarian function.Conclusions(1)Metformin can improve the cell viability of oxidative-damaged cells,reduce reactive oxygen species,increase mitochondrial membrane potential,reduce apoptosis and improve oxidoreductase activity.Metformin can protect cells from oxidative damage by activating AMPK and regulating autophagy homeostasis.(2)Metformin promotes the expression of TFEB and regulates autophagy through AMPK.There is interaction between AMPK and TFEB,which may be one of the factors that AMPK regulates TFEB.Metformin can activate TFEB by promoting nuclear translocation of TFEB and play a protective role in cells.(3)Metformin can improve the reduction of lysosome number and function induced by oxidative damage,which is related to enhancing the binding of TFEB to LAMP1 promoter region and promoting LAMP1 transcription.(4)Metformin can improve D-gal-induced ovarian dysfunction in POI mice,at least partially through AMPK/TFEB/LAMP1 axis to protect ovarian function.(5)Metformin can enhance the antioxidant damage of ovarian granulosa cells and mouse ovary,at least partly by activating AMPK,up-regulating TFEB expression and promoting LAMP1 gene transcription,thus enhancing lysosomal function,reducing autophagy accumulation,restoring autophagic flux and exerting cell protection.