| Purposes:Network pharmacology methods,experiments of in vivo and in vitro were used to explore the signal pathways and target mechanisms related to Qiangjing Pills in the treatment of late-onset hypogonadism,and verify whether Qiangjing Pills can regulate androgen and senescence-related ROS/FOXO4-p53 signals in LOH model rats and senescent Leydig cells.Methods:Chapter 1:Network pharmacology methods is used to query the potential targets of the effective ingredients of Qiangjing Pills and potential targets of LOH disease through the database.Cytoscape software is used to construct the"drug-target-disease"protein interaction network of Qiangjing Pills for the treatment of LOH,and screen the core targets and direct targets.These methods have been used to explore the potential signal pathways and target mechanisms of Qiangjing Pills in the treatment of LOH.Chapter 2:Set up a 2-month-old youth rat control and a 19-month-old LOH rat model,with Qiangjing Pills as intervention measures,and testosterone propionate injection as the positive control.To detect the serum total testosterone（TT）,free testosterone（FT）,luteinizing hormone（LH）,serum sex hormone binding globulin（SHBG）,testicular tissue androgen receptor（AR）,and the protein expression levels of St AR,3β-HSD,CYP11A1,CYP17A1 in testosterone synthesis pathway in LOH model rats,testicular tissue pathology,and calculate the testosterone secretion index（TSI）of rats,study of the effect of Qiangjing Pills on the production and utilization of testosterone in LOH model rats.Chapter 3:Using ELISA method to detect ROS,manganese-superoxide dismutase（SOD2）and catalase（CAT）in testis tissue of LOH model rats.Using Western Blot to detect the protein expression of aging-related FOXO4-p53 axis in testis tissue of LOH model rats.To study the effect of Qiangjing Pills on oxidative stress and ROS/FOXO4-p53 signal in testis tissue of LOH model rats.Chapter 4:The Drug-containing serum of Qiangjing pills will be made,H2O2-induced senescent Leydig cell model will be constructed,and the senescent Leydig cells will be stimulated by LH to simulate the functional compensation of testosterone secretion in senescent Leydig cells.Low,medium and high doses of medicated serum will be used to intervene in aging Leydig cells,and ROS scavenger NAC will be used as a positive control.ELISA will be used to detect oxidative stress and agingβ-galactosidase activity.Flow cytometry will be used to detect cell apoptosis and cell cycle and Western Blot to detect the protein expression of testosterone synthesis pathway and FOXO4-p53 axin.Immunofluorescence will be used to detect the nuclei transposition of FOXO4 and phosphorylated p53.Observe the effects of Qiangjing Pills on the secretion of testosterone,senescence state and ROS/FOXO4-p53 signal in aging Leydig cells.Results:Chapter 1:Using network pharmacology methods to explore the potential signal pathways and target mechanisms of Qiangjing Pills in the treatment of LOHA total of 128 potential active ingredients of Qiangjing Pills and 183 potential targets were discovered through network pharmacology methods.A total of 171 LOH disease-related targets were discovered.Qiangjing Pills and LOH diseases share 26 targets,including AR and p53.By constructing a drug-target-disease protein interaction network for Qiangjing Pills to treat LOH,and using an eight-fold median to screen core targets,a total of 154 potential core targets for Qiangjing Pills to interfere with LOH were obtained.The potential signal pathway were analyzed through DAVID database.The top 25 signal pathways of Qiangjing Pills in the treatment of LOH,including cell cycle,cell aging,FOXO signaling pathway,p53 signaling pathway,etc.In the cellular senescence signal pathway,many factors such as ROS and oxidative stress can affect cell senescence by acting on FOXOs and p53.This study will verify whether the mechanism of Qiangjing Pills in the treatment of LOH is related to AR,FOXOs,and p53 signals.Chapter 2:Effect of Qiangjing Pills on androgen production and utilization in LOH model rats（1）The effect of Qiangjing Pills on the secretion index of testosterone and serum sex hormones in rats.（1）Testosterone secretion index（TSI）:TSI of the model group was significantly lower than that of the young control group（P<0.05）,the medium and high doses of Qiangjing Pills significantly reduced the TSI of the model rats（P<0.05）;the testosterone propionate group maintained the TSI of the model rats（P>0.05）.Compared with the testosterone propionate group,there was no significant difference in TSI between each dose group of Qiangjing Pills（P>0.05）.It showed that the 19-month-old male SD rats conform to the subtype of the LOH rat model-the compensatory type.The medium and high doses of Qiangjing Pills have a significant inhibitory effect on the testosterone secretion function of compensated LOH model rats,which is beneficial to alleviate the compensatory secretion of testosterone in aging Leydig cells and reduce the burden of testosterone secretion in aging Leydig cells.（2）Serum LH,FT,SHBG,TT:There was no significant difference in LH,FT,SHBG,TT between the19-month-old model group and the 2-month-old young control group（P>0.05）.After the intervention of Qiangjing Pills,the LH,FT,and SHBG of 19-month-old SD rats showed an increasing trend（P>0.05）,and TT showed a decreasing trend（P>0.05）.Testosterone propionate significantly increased the LH and TT of 19-month-old SD rats（P<0.05）;the TT of the testosterone propionate group was significantly higher than that of the Qiangjing Pill group（P<0.05）.Qiangjing Pills increased the levels of LH,FT and SHBG in compensated LOH model rats to a certain extent,and maintained the level of serum TT.（2）The effect of Qiangjing Pills on the expression of AR protein in the testis of compensated LOH model rats.AR expression in the model group was significantly up-regulated than the young group（P<0.05）.Qiangjing Pills and testosterone propionate significantly up-regulated AR protein expression in model rats（P<0.05 or P<0.01）;testosterone propionate up-regulated AR protein expression stronger than Qiangjing Pills（P<0.01）.Qiangjing Pills can significantly up-regulate the expression of AR protein in the testis of LOH model rats,which is beneficial to increase the availability of androgen in the testis tissue of LOH model rats,but its effect is not as obvious as that of testosterone propionate.（3）The effect of Qiangjing Pills on the expression of St AR,3β-HSD,CYP11A1,CYP17A1 protein in the testis of the compensated LOH model rats.The expression of St AR and 3β-HSD protein in the model group was significantly higher than that of the young control group（P<0.01）.The protein expression of CYP11A1 and CYP17A1was not statistically different from that of young controls（P>0.05）.Each dose of Qiangjing Pills and testosterone propionate significantly down-regulated the expression of St AR,3β-HSD,and CYP11A1 protein in compensated LOH model rats（P<0.05 or P<0.01）.And the testicular St AR,3β-HSD,and CYP11A1 protein expressions in each dose of Qiangjing pills were higher than those in the testosterone propionate group（P<0.01）.The medium and high doses of Qiangjing pills and testosterone propionate significantly up-regulated the expression of CYP17A1 protein in compensated LOH model rats（P<0.05 or P<0.01）;and the medium dose of Qiangjing pills increased the expression of CYP17A1 protein stronger than testosterone propionate（P<0.01）.It shows that Qiangjing pills and testosterone propionate significantly inhibit the expression of St AR,3β-HSD and CYP11A1 protein in the testis of compensated LOH model rats,and the inhibitory effect of testosterone propionate is more obvious than that of Qiangjing pills.Qiangjing pills and testosterone propionate up-regulated the expression of CYP17A1 protein in the testis of LOH model rats,and the effect of medium dose of Qiangjing pills was stronger than that of testosterone propionate.Chapter 3:Effect of Qiangjing Pills on ROS/FOXO4-p53 Signal in Testis Tissue of Compensated LOH Model Rats（1）The effect of Qiangjing Pills on ROS,SOD2 and CAT in the testis tissues of compensated LOH model rats.Qiangjing Pills have a tendency to decrease testicular ROS in LOH model rats,and the high-dose group significantly reduced testicular ROS（P<0.05）.Qiangjing Pills slightly reduced SOD2 and CAT in the testis of rats（P>0.05）.Testosterone propionate significantly reduced ROS and CAT in rat testis tissue（P<0.05）.（2）The effect of Qiangjing Pills on the protein expression of FOXO4-p53 signal in the testis of compensated LOH model rats.The expression of FOXO4 protein in the testis of compensated LOH model rats is the same as that of 2-month-old young control rats（P>0.05）,but p53,phosphorylated p53,p21 senescence protein increased（P<0.01）.Qiangjing Pills and testosterone propionate significantly down-regulated FOXO4,p53,phosphorylated p53,and p21 protein expression in the testis of LOH model rats,and the effect of testosterone propionate was stronger than that of Qiangjing Pills.Chapter 4:Effects of drug-containing serum of Qiangjing pills on testosterone secretion and senescent ROS/FOXO4-p53 signal of senescent Leydig cells.（1）The effect of drug-containing serum of Qiangjing pills on testosterone secretion of aging Leydig cells.（1）The testosterone secretion function of senescent Leydig cells was significantly lower than that of normal cells（P<0.05）,and the drug-containing serum of Qiangjing pills could not improve the testosterone secretion of senescent Leydig cells（P>0.05）.NAC improved testosterone secretion to a certain extent（P>0.05）.（2）The effect of drug-containing serum of Qiangjing pills on the senescence state of functionally compensated senescent Leydig cells.When exogenous LH is used to stimulate functional compensation of senescent Leydig cells:（1）The drug-containing serum of Qiangjing pills increasedβ-galactosidase activity of senescent Leydig cells to a certain extent（P>0.05）,while NAC did not increaseβ-galactosidase activity of senescent Leydig cells.（2）Apoptosis:The drug-containing serum of Qiangjing pills had no effect on apoptosis of senescent Leydig cells.（3）Cell cycle:Compared with normal cells,the proportion of senescent Leydig cells in G1phase is reduced,while the proportion of cells in S phase is increased（P<0.01）.Qiangjing Pills containing serum can aggravate S phase cell cycle arrest.（3）The effect of drug-containing serum of Qiangjing pills on testosterone secretion of functionally compensated senescent Leydig cells.When exogenous LH was used to stimulate functional compensation of senescent Leydig cells:（1）The drug-containing serum of Qiangjing pills and NAC could promote testosterone secretion of functionally compensated senescent Leydig cells to a certain extent（P>0.05）.（2）The effect of drug-containing serum of Qiangjing pills on testosterone synthesis pathway:The Drug-containing serum of Qiangjing pills could significantly increase the compensatory expression of CYP11A1 protein;its effect was stronger than NAC（P<0.01）.The medicated serum of medium and high dose of Qiangjing Pills increased the compensatory expression of St AR protein（P>0.05）.The medium and high doses of drug-containing serum of Qiangjing pills and NAC both increased the protein expression of CYP11A1 to a certain extent（P>0.05）.（4）The effect of drug-containing serum of Qiangjing pills on ROS/FOXO4-p53signal of functionally compensated aging Leydig cells.When exogenous LH was used to stimulate functional compensation of senescent Leydig cells:（1）The drug-containing serum of Qiangjing pills increased the ROS level of functionally compensated senescent Leydig cells to a certain extent（P>0.05）,and increased the level of cell CAT（P>0.05）),and increased oxidative stress to a certain extent.NAC,a ROS scavenger,did not increase ROS and CAT in senescent Leydig cells.（2）The expression of FOXO4 in senescent Leydig cells was significantly higher than that in young cells.The drug-containing serum of Qiangjing pills maintained the functional compensation of FOXO4 protein expression in senescent Leydig cells（P>0.05）,while NAC significantly up-regulated FOXO4 protein expression（P<0.01）.The p53 protein of old Leydig cells was significantly increased than that of young cells（P<0.01）;the drug-containing serum of Qiangjing pills significantly increased the expression of p53 protein in functionally compensated senescent Leydig cells,while NAC inhibited the expression of p53 protein.Qiangjing Pills and NAC enhanced the transcriptional activity of FOXO4 and p-p53 in senescent Leydig cells to a certain extent.Conclusion:Mechanism prediction:1.The direct targets of Qiangjing pills to treat LOH include AR and p53,and the signal pathways involved include FOXO signal,p53 signal,etc.In vivo:1.In vivo,Qiangjing Pills can improve androgen utilization by up-regulating AR expression in the testis tissues and serum FT of compensated LOH model rats;and Qiangjing Pills can inhibit the protein expression of St AR,3β-HSD,CYP11A1,up-regulate the protein expression of CYP17A1 in testosterone synthesis pathway in the testis tissues of compensated LOH model rats,alleviate the compensation of testosterone secretion,and maintain the level of circulating TT.2.The intervention of Qiangjing Pills reduced the ROS and inhibited the FOXO4-p53signal related to aging in the testis tissue of the compensated LOH model rat.In vitro:1.In vitro,the drug-containing serum of Qiangjing pills can not affect the testosterone secretion of aging Leydig cells.2.Under the condition of exogenous LH stimulation at a concentration of 6.5 m IU/m L to simulate the functional compensation of aging Leydig cells,the drug-containing serum of Qiangjing Pills can up-regulate the compensatory expression of St AR and CYP11A1 in aging Leydig cells,increase the testosterone secretion and the functional compensation of senescent Leydig cells.3.Under the stimulation of exogenous LH at a concentration of 6.5 m IU/m L,the drug-containing serum of Qiangjing Pills can promote functionally compensated senescent Leydig cells to secrete testosterone,result in more ROS production,enhance senescence-related ROS/FOXO4-p53 signals,increase cellsβ-galactosidase activity and aggravate cell cycle arrest,and ultimately aggravate the senescence of functionally compensated Leydig cells.Summary:In vitro,under normal circumstances,the drug-containing serum of Qiangjing Pills can not increase the burden of testosterone secretion in aging Leydig cells.Under the condition of simulating the compensatory secretion of testosterone in aging Leydig cells,the drug-containing serum of Qiangjing Pills can promote the secretion of testosterone in aging Leydig cells to a certain extent,but this will increase the production of ROS and up-regulate the senescence-related ROS/FOXO4-p53 signal,which further aggravates Senescence of Leydig cells.In vivo,Qiangjing Pills increase the availability of androgen by up-regulating the testis AR protein and serum FT levels of compensated LOH model rats,which is beneficial to reduce the burden of testosterone secretion in Leydig cells.At the same time,Qiangjing Pills inhibits the abnormal increase of St AR,3β-HSD,and CYP11A1 proteins in the testicular testosterone synthesis pathway,reduces ROS and testicular oxidative stress levels,thereby down-regulating testicular aging ROS/FOXO4-p53 signals.Thereby,it has an anti-aging effect on the testis and Leydig cells of the compensated LOH model rats.All in all:In vitro,promoting Leydig cells to secrete testosterone will aggravate Leydig cell senescence.In vivo,increasing the androgen availability of compensated LOH is beneficial to alleviate the burden of Leydig cells and has a protective effect on LOH. |
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