Effects Of Liuwei Dihuang Pill On The Improvement Of Chronic Testicular Inflammation In Sub-acute Aging Rats By AMPK/SIRT1/NF-κB Pathway

Objective To study effects of Liuwei Dihuang Pill（LP）on D-galactose induced chronic inflammation of testis in subacute-aging rats,and to explore the possible mechanism of the intervention of LP in testis aging,so as to provide a scientific basis for the clinical use of LP in the treatment of testis inflammatory aging.Methods 30 SPF 8-week-old SD rats were randomly divided into control group,aging model group and LP group,with 10 rats in each group.In the control group,normal saline was injected subcutaneously every day for 8 weeks.From the 5th week,normal saline was gavaged daily for 4 weeks while subcutaneously injected.In the aging model group,D-galactose solution was subcutaneously injected at 125mg/kg daily for 8 weeks.From the 5th week,normal saline was gavaged daily while subcutaneously injected.In LP group,D-galactose was given subcutaneous injection every day for 8 weeks.From the 5th week,LP solution was given intragastric gavage at 0.9g/kg at the same time of subcutaneous injection.8 weeks later,testis of rats was taken and pathological changes was observed by HE staining.Western blotting was used to detect the protein expression levels of p16^INK4A,p21^WAF1/CIP1,AMPK,p-AMPK,SIRT1,NF-κB,p-p65,TNF-α,IL-1β,IL-6,IL-10 and HO-1 in testis tissues of rats.The expression ofβ-galactosidase,SIRT1,NF-κB,P-p65,TNF-α,IL-1β,IL-6,IL-10 and HO-1 in testis were detected by immunohistochemical staining.The experimental data were processed by SPSS22.0 software andα=0.05 was taken as the test level.Results1.Model construction and evaluation:Compared with the blank control group,HE results showed that the testicular tissue in the aging model group was significantly atrophied and a large number of spermatogenic epithelial cells were shed.Western blotting results showed that the protein expression levels of P16^INK4A and P21^WAF1/CIP1 significantly increased,and the difference was statistically significant.Immunohistochemical results presented that the opposite trend of localization ofβ-galactosidase protein.2.Evaluation of LP on testicular aging level of aging rat model:Compared with the aging model group,HE results showed that the germ cells in all layers of testicular tissue of rats in the LP group were arranged closely and orderly without obvious atrophy.Western blotting results showed that the protein expression levels of P16^INK4A and P21^WAF1/CIP1 significantly decreased,and the difference was statistically significant.Immunohistochemical results showed that the localization ofβ-galactosidase protein showed an opposite trend.3.Effects of LP on inflammatory pathway and related protein expression:Compared with the aging model group,Western blotting results showed that the protein expression levels of TNF-α,IL-1βand IL-6 in testis tissues of LP group significantly decreased,the protein expression levels of SIRT1,IL-10 and HO-1 significantly increased,and the p-AMPK/AMPK ratio was increased,but P-p65/P65 ratio was decreased.Immunohistochemical results showed that the same trend in localization of NF-κB,TNF-α,IL-1βand IL-6 protein,while the localization of SIRT1,IL-10 and HO-1 protein showed an opposite trend.Conclusion LP alleviates testicular inflammation in aging rats by inhibiting NF-κB activity,whose mechanism may be related to the activation of AMPK/SIRT1 pathway.