| Objective The purpose of this study was to explore the effects of Lycium barbarum seed oil(LBSO)on the anti-oxidantion of testis and mouse support cells(TM4 cells).It was further corroborated that LBSO activated SIRT3 to improve the tolerance of TM4 cells in the intervention of D-galactose(D-gal),alleviating oxidative stress of mitochondria of testis and TM4 cells.Effects of LBSO on anti-oxidation were studied in order to provide a new approach to protect testis against aging and improve the function of testis of male.Methods In vivo experiments:60 8-week-old male SD rats were randomly divided into 6 groups,including control group,aging model group,very low dose group,low dose group,medium dose group and high dose group.Except for control group,subacute aging model was established in other groups,and subcutaneous injection of D-gal at 125 mg/kg/d was given for 8 weeks,while the same amount of normal saline was given subcutaneous injection in control group.4 weeks later in subcutaneous injection,low dose group was gavaged by LBSO at 500 mg/kg/d,low dose group gavaged by LBSO at 1000 mg/kg/d,middle dose group gavaged by LBSO at 1500 mg/kg/d,high dose group gavaged by LBSO at 2000 mg/kg/d for 4 weeks,and control group and aging model group give the same amount of saline lavage for 4 weeks,at the same time continue to be injected subcutaneously by D-gal until 8 weeks.The establishment of sub-acutely aging model was evaluated by observation of testicular histological changes,immunohistochemical results of galactosidase and aging related proteins(p16INK4A,p21WAF1/CIP1,γH2A.X protein and galactosidase).The serum levels of related oxidation factors,such as superoxide dismutase(SOD),glutathione(GSH),reactive oxygen species(ROS),malondialdehyde(MDA)and 8-hydroxy-deoxyguanosine(8-OHDG);the protein expression levels of Nrf2,SIRT3,HO-1,SOD-1 and SOD-2 in testis were detected.The testicular morphology was observed,as well as the spermatogenic function of testis was assessed by Johnsen score,and the secretion function of testis was detected by serum levels of testosterone and serum inhibitory B(INHB).In addition,SIRT3 localization and expression in testicular,especially expression in Sertoli cells,was observed by immunofluorescence co-localization.In vitro experiment:Different concentrations of D-gal were used to stimulate TM4 cells to establish the aging model of TM4 cells.The cells were divided into 5 groups,which were treated with complete medium or 4 different concentrations of D-galactose at 50 mmol/L,100 mmol/L,150 mmol/L and 200 mmol/L for 24 h,36 h and 48 h,respectively.Cell inhibition rate by CCK-8,galactosidase staining and aging protein expression levels were used to screen out optimal concentration and time of D-gal.On this basis,5 groups were set up,including aging model group and 4 groups with different concentration of LBSO experimental groups(25 μg/mL,50 μg/mL,100μg/mL,150 μg/mL).Except for aging model group,different LBSO was premeditated for 6 h,and then 200 mmol/L D-gal was added for 48 h.Cell viability of each group was detected by CCK-8.The protein expression levels of NRF2,SIRT3,HO-1,SOD-1 and SOD-2 were detected.In order to investigate the anti-oxidant mechanism of LBSO,SIRT3 was silenced by lentivirus,and four groups were set up:senility group,Lycium barbarum seed oil group,SIRT3 silencing group(si-SIRT3)and si-SIRT3+LBSO group.LBSO group was treated with 100 μg/mL LBSO for 6 h in advance and then D-gal was added for 66 h.In si-SIRT3 group,si-SIRT3 lentivirus was pre-transfected into TM4 cells for 6 h and D-gal was added for 66 h.Si-SIRT3+LBSO group:LBSO and Si-SIRT3 lentivirus interfered with TM4 cells for 6 h in advance,and D-gal was added for 66 h.The aging group TM4 cells were added with D-gal at the same time as other experiments.The expression levels of NRF2,SIRT3,HO-1,SOD-1 and SOD-2 were detected.In addition,lentivirus overexpression of SIRT3 was divided into three groups:control group,LBSO group and SIRT3 overexpression group(OE-SIRT3 group).The expression of SIRT3,p-AMPK,AMPK,p-PGC-1α and PGC-1α in TM4 cells were detected.Results 1.Evaluation of subacute aging rat model:Compared to control group,anti-oxidant factors such as SOD and GSH were significantly decreased,ROS,MDA and 8-OHDG oxides were significantly increased in serum of aging model rats,and the difference was statistically significant.The expression of aging proteins P16INK4A,P21WAF1/CIP1,γH2A.X protein and galactosidase were significantly up-regulated in aging model group,as comapered to control group.Compared to control group,in the aging model group,the number of supporting cells and germ cells at all levels of testicular tissue decreased significantly,and the normal arrangement from spermatogonia to spermatozoa cells was lost.β-galactosidase staining showed significantly higher expression in senile testis.2.LBSO improved oxidative stress injury of testis in sub-acutely aging rats:Compared to aging model group,except for the very low group,anti-oxidant factors of SOD and GSH in serum of rats in different concentrations of LBSO groups were significantly increased,and key oxidative factors such as ROS,MDA and 8-OHDG were significantly decreased,but there was no statistical difference between the low,middle and high dose groups.At the same time,the expressions of HO-1,NRF2,SOD-1 and SOD-2 in testis of rats in the three groups of low,middle and high Lycium berry seed oil groups increased significantly,but there was no statistical difference between the three groups,and there was no dependence of concentration gradient.3.LBSO activates SIRT3 expression in rat testis:After intragastric administration of LBSO in aging rats,the mRNA and protein expression levels of SIRT3 in rat testis were significantly up-regulated,and the expression of SIRT3 was mainly concentrated in testicular sertoli cells.4.LBSO improved testicular function in rats:Johnsen score was used to evaluate the spermatogenic function of testicular tissue.Compared to the aging model,testicular tissue score was significantly increased from 7.69±0.24 to 8.85±0.19 after intervention with LBSO.Meanwhile the levels of INHB and testosterone secreted by serum supporting cells in LBSO group were significantly higher than those in aging model group.5.TM4 cell aging model evaluation:compared to control group,the inhibition rate of TM4 cells by D-gal intervention for 24 h and 36 h is negative,the cell vitality but there is a growing trend.However,when the the TM4 cells was intervened by D-gal with 200 mmol/L for 48 h,the proliferation of that was significantly inhibited.While the 200 mmol/L concentration of D-gal intervention,galactosidase staining positive cell rate significantly increased;the expression of aging related proteins was significantly up-regulated in senescence model group.6.Effects of LBSO on anti-oxiadation of aging TM4 cells:the number and viability of aging TM4 cells were significantly increased and the positive rate of galactosidase staining was also significantly decreased after the intervention of LB SO at concentrations of 100 μg/mL and 150 μg/mL;The expressions of NRF2,SIRT3,HO-1,SOD-1 and SOD-2 in TM4 cells were significantly up-regulated after LBSO intervention.7.LBSO activated SIRT3 to protect TM4 from oxidative stress:levels of mRNA and protein SIRT3 were upregulated in TM4 cells in LBSO group,compared to control group.Immunofluorescence results showed that SIRT3 localization expression was upregulated in TM4 cells;The expression of SIRT3,HO-1,SOD-1 and SOD-2 in TM4 cells decreased significantly after SIRT3 silencing.Compared to aging model group,expressions of SIRT3,p-AMPK/AMPK and p-PGC-1α/PGC-1α were significantly up-regulated in SIRT3 overexpression group and LBSO group.Conclusion 1.Based on the establishment of D-gal induced sub-acutely aging model,LBSO could protect the testis of aging rats from oxidative stress and improve the function of testis of aging rats by activating SIRT3.2.The aging model of TM4 cells was successfully established by 200 mmol/L gal for 48h.On this basis,LBSO interfered with TM4 aging cells,and effects of LBSO on anti-oxidation to protect TM4 cells from oxidative injury by activating SIRT3 were clarified.3.LBSO activated TM4 cells to express SIRT3,activating AMPK/PGC-1α pathway to stabilize mitochondrial homeostasis and protect cells from oxidative stress injury. |
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