| Asthenospermia is one of the common types of male infertility.Its high incidence rate is one of the main causes of male fertility decline and male infertility.Traditional Chinese medicine has a long history in the treatment of asthenospermia,which is safe and effective.More and more patients begin to seek traditional Chinese medicine treatment.Qixiong formula(qxf)is an empirical formula of the Department of andrology,Xiyuan Hospital,Chinese Academy of traditional Chinese medicine.It has the effects of strengthening the spleen and kidney,promoting blood circulation and generating sperm.Previous clinical studies have confirmed that Qixiong formula can effectively improve the percentage of forward movement(RP)spermatozoa(15.98%±8.01%,22.71%± 9.00%)and the percentage of PR+NP spermatozoa(29.29%± 12.20%,38.71%± 14.06%)in patients with asthenospermia at the 8th and 12th weeks,Compared with that before treatment(PR:9.47%± 6.23%,PR+NP:18.42%± 8.72%)(P<0.05),and at 12 weeks of treatment,the effect of Qixiong formula on improving PR+NP grade sperm is better than that of zuokanitine oral liquid(26.17%± 15.40%),but its specific treatment mechanism is not clear.This study intends to predict the possible mechanism of Qixiong formula in the treatment of asthenospermia on the basis of early clinical practice.Then the animal experiment further confirmed the effectiveness and safety of Qixiong formula in the treatment of asthenospermia rats,and verified its mechanism of action.Part 1 Mechanism based on network pharmacology for the treatment of asthenozoospermia by Qixiong formulaObjectiveTo study the network pharmacology of Qixiong formula and explore the mechanism of Qixiong formula in the treatment of oligoasthenospermia.MethodsBy screening the effective active components and action targets of Qixiong formula,search the targets related to asthenospermia,obtain the intersection targets,construct the protein interaction network to screen the key targets,visualize the drug active component target interaction relationship,enrich and analyze the intersection targets,and verify the molecular docking between the core components and targets.Results1.122 main active components and 270 related targets of qxf meeting the screening conditions were obtained,and 3224 related targets of asthenospermia were obtained.162 Qixiong formula were obtained after intersection,which can be used to treat asthenospermia.2.The results of GO enrichment analysis showed that Qixiong formula treatment of asthenospermia may occur in membrane raft,receptor complex,transcription regulatory complex,protein kinase complex,mitochondrial matrix and other parts.It plays a role by regulating the reaction of organic ring compounds,cell movement,apoptosis,protein phosphorylation and other ways,and is closely related to transcription factors,protein kinase,protein phosphatase and so on.The results of KEGG enrichment analysis suggest that Qixiong formula treatment of asthenospermia may involve multiple signal pathways such as PI3K/Akt、MAPK、HIF-1、FoxO and so on.3.The results of molecular docking suggest that the main active components of Qixiong formula have strong binding force with the core target.ConclusionsThis study reflects the characteristics of multi-component-multi-targetmulti-pathway of Qixiong formula,combined with the literature,it is predicted that Qixiong formula may play a role in the treatment of asthenospermia through pi3k/akt/mtor signal pathway,and provides a clear direction for further research on the mechanism of Qixiong formula in the treatment of asthenospermia.Part 2 Study on the mechanism of Qixiong recipe in the treatment of asthenospermia model rats based on PI3K/Akt/mTOR autophagy pathwayObjectiveThrough animal experiments,the mechanism of Qixiong formula in the treatment of asthenospermia will be studied from the perspective of autophagy mediated by PI3K/Akt/mTOR signal pathway.MethodsSixty rats were randomly divided into blank group,model group,low dose group,medium dose group,high dose group and L-carnitine group.The blank group was given lm/100g 0.5%sodium carboxymethyl cellulose(CMC-Na)solution;the model group was given 0.9%NaCl solution+400mg/(kg·d)Ornidazole solution(ORN);the low,medium and high dose groups of Qixiong formula were given 5.79g,11.59g,23.18g crude drug/kg and 400mg/(kg·d)ORN respectively,and the levocarnitine group was given levocarnitine oral liquid 100mg/(kg·d)+400mg/((kg·d)ORN.ORN suspension was given to model group,low dose group,medium dose group and high dose group from 8:00 to 9:00 every morning;from 5:00 to 6:00 p.m.every day,the low-dose group,medium dose group and high-dose group were given Qixiong formula concentrated solution,and the levocarnitine group was given levocarnitine oral solution.The dose of lml/100g body weight was continuously administered by gavage for 28 days.After the 28 days,the group was anesthetized,and weighed,blood was taken from abdominal aorta,bilateral testis and epididymis were taken,and epididymis and testicular index were calculated.Epididymal homogenate was prepared from the left epididymal tail,and sperm motility was evaluated in a warm bath.The right epididymis of rats was cut longitudinally,and the testis was routinely sectioned along the equatorial plane for the histomorphological observation of testis and epididymis.Sex hormones(FSH,LH,T)were measured by ELISA,and liver function(ALT,AST)and renal function(UREA,BUN,CREA)were measured by automatic biochemical analyzer.The expression of PI3K,p-PI3K,Akt,p-Akt,mTOR,p-mTOR,p62,beclin-1 and LC3Ⅱ/Ⅰ in epididymis of rats was detected by Western blot,and the expression of PI3K,p-PI3K,Akt,p-Akt,p-mTOR,p62,beclin-1 and LC3Ⅱ/Ⅰin epididymis of rats was detected by immunohistochemistry.Results1.General condition of rats:on the 9th day of the experiment,rats in the low-dose group died one;On the 20th day of the experiment,one rat died in the high-dose group,and no rat death occurred in the blank,model,and medium-and levocarnitine groups.In the blank group,the diet and drinking of rats were not significantly changed,their body hair was thick and shiny,their mental status was good,their activity amount was large,there was a cage fighting phenomenon,and their stool condition was normal.In the model group,the rats had decreased amount of diet,loose body hair,glossiness,burnout,some rats were not active in locomotion,the fight phenomenon was reduced,their stool color was lighter,their quality was thin,and their stool color was normal.The rats in the low-dose group of Qixiong formula had loose but shiny body hair,some hair oils and fats were rich,their mental status was better than that of the model group,some were not motile,there was cage fighting phenomenon,or appeared irritable,their stools were occasional with thin stools,and their stools were normal in color;The mental status and stools improved in the middle and high dosage groups of Qixiong formula compared with those in the lower dosage group.The diet of the levocarnitine group did not change significantly,hair oil was abundant,mental status was better than that of the model group,there was a cage fighting phenomenon,stools were occasional with thin quality,and the color of the stools was normal.2.Changes of body weight,testis and epididymis weight and index:there was no significant difference in body weight among groups(P>0.05).In terms of testicular weight,there was no significant difference in testicular weight among groups(P>0.05).In terms of epididymis weight,the epididymis weight of the model group was lower than that of the blank group,and the difference was statistically significant(P<0.01).Compared with the model group,the weight of epididymis in the high-dose group and L-carnitine group was significantly higher than that in the model group(P<0.01),the weight of epididymis in the medium-dose group was significantly higher than that in the model group(P<0.05),and there was no significant difference between the low-dose group and the model group.In terms of testicular index,there was no significant difference between groups(P>0.05).In terms of epididymal index,the epididymal index of the model group was significantly lower than that of the blank group(P<0.01).Compared with the model group,the epididymal index of high-dose group and levocarnitine group increased,the difference was statistically significant(P<0.05),and the epididymal index of low and medium dose groups increased,the difference was not statistically significant(P>0.05).3.Changes of semen parameters:compared with the blank group,there was no significant change in sperm concentration in the model group(P>0.05),and the percentage of PR grade sperm and sperm motility decreased significantly(P<0.01).Compared with the model group,there was no significant difference in the changes of sperm concentration in low,medium and high dose groups of Qixiong formula and levocarnitine group(P>0.05).There was no significant difference in the percentage and activity rate of PR grade sperm in the low dose group of Qixiong formula,the percentage and activity rate of PR grade sperm in the medium dose group of Qixiong formula increased(P<0.05),and the percentage and activity rate of PR grade sperm in the high dose group of Qixiong formula and levocarnitine group increased significantly(P<0.01).There was no significant difference in the percentage and activity rate of PR grade sperm in the high dose group of Qixiong formula compared with Levocarnitine group.4.In terms of liver function,compared with the blank group,there was no significant difference in the changes of serum liver function indexes(ALT and AST)in the model group(P>0.05).Compared with the model group,there was no significant difference in the levels of ALT and AST in serum of rats in low,medium and high dose groups of Qixiong formula and levocarnitine group(P>0.05).In terms of renal function,compared with the blank group,there was no significant difference in the changes of serum renal function indexes(UREA,BUN,CREA)in the model group(P>0.05).Compared with the model group,there was no significant difference in the levels of serum renal function indexes(UREA,BUN,CREA)in low,medium and high dose groups of Qixiong formula and levocarnitine groups(P>0.05).5.Changes of sex hormones:there was no significant difference in the levels of serum FSH,LH and T in each group(P>0.05).6.Histomorphological changes of rat testis and epididymis:ornidazole has no significant effect on the morphology of rat testis after modeling,and Qixiong formula and levocarnitine groups have no significant effect on the morphology of rat testis.Staining of epididymal sections:in the blank group,the epididymal tubes are arranged regularly and orderly,the lumen size is the same,the gap is normal,the glandular cavity has no expansion,the cells are arranged closely and the size is the same,there is no degeneration or necrosis,and a large number of linear sperm can be seen in the lumen.In the model group,the lumen is arranged disorderly and disorderly,the glands are slightly reduced,the lumen size is inconsistent,the gap is increased,the tube wall is thinner,the vacuoles in the cytoplasm are denatured,the vacuoles are increased,the sperm is reduced or absent,and the cell fragments and eosinophils are deposited.The lumens in the low and medium dose groups were arranged in a roughly regular and orderly manner,with inconsistent sizes,and some lumen gaps increased.In the low dose group,some tube walls became thinner,with occasional vacuolar degeneration in the cytoplasm,reduction or absence of sperm,and deposition of cell fragments and eosinophils;compared with the model group,the tube wall of the medium dose group was thicker,the vacuoles in the cytoplasm were reduced,a large number of linear sperm and eosinophilic material deposition were seen in the lumen.In the high-dose group,the lumen is arranged regularly and orderly,the size is relatively consistent,the lumen space is normal,the cells are arranged closely,the size is consistent,no degeneration or necrosis is found,and a large number of linear sperm can be seen in the lumen.In the left carnitine group,the lumen is roughly regular,the size is relatively consistent,the lumen space is normal,the cells are closely arranged,and a large number of linear sperm can be seen in the lumen.7.There was no significant difference between the model group and the control group(P<0.05).Compared with the model group,the IOD values measured by p-PI3K,p-Akt and p-mTOR in epididymis of Qixiong formula group were increased(P<0.05).Compared with the blank group,the IOD values measured by autophagy protein LC3 and Beclinl in epididymal tissue of the model group were significantly increased(P<0.01),and the IOD values measured by p62 were significantly decreased(P<0.01).Compared with the model group,the IOD values measured by LC3 in epididymal tissue of Qixiong formula group were all decreased(P<0.05),Beclinl in low-dose group was lower than that in model group(P<0.05),and Beclinl in medium and high-dose groups was significantly lower than that in model group(P<0.01).The IOD value measured by p62 in epididymis of Qixiong formula group increased(P<0.05).8.Expression changes of PI3K/Akt/mTOR signal pathway related proteins:compared with the blank group,there was no significant difference in the expression of PI3K,Akt and mTOR in epididymal tissue in the model group.Compared with the model group,there was no significant change in the expression of PI3K,Akt and mTOR in each dose group of Qixiong formula.Compared with the blank group,the expression of epididymal tissue proteins p-PI3K,p-Akt and p-mTOR in the model group decreased significantly(P<0.01).The epididymal tissue proteins p-PI3K,p-Akt and p-mTOR in the low-dose group increased compared with the model group,but the difference was not statistically significant(P>0.05).The expression of epididymal tissue protein p-PI3K and p-Akt in the medium dose group was significantly higher than that in the model group(P<0.05).The expression of epididymal tissue protein p-mTOR in the medium dose group was not significantly higher than that in the model group(P>0.05);epididymal tissue proteins p-PI3K,p-Akt and p-mTOR in the high-dose group were significantly higher than those in the model group(P<0.01).9.Changes of autophagy related protein expression level:compared with the blank group,the expression levels of beclin-1 as well as LC3II/I in epididymal tissue in the model group were significantly increased,and the differences were statistically significant(P<0.01);the expression of p62 decreased compared with the blank group(P<0.05).Compared with the model group,beclin-1 expression decreased in the low-dose group of Qixiong formula(P<0.05),and the relative expression of LC3Ⅱ/Ⅰ decreased,and the differences were not statistically significant(P>0.05);the relative expression of beclin-1 and LC3Ⅱ/Ⅰ decreased significantly in the medium and high dosage groups,with significant differences(P<0.01);the relative expression of p62 in low-dose histones was increased by Qixiong formula,but the difference was not statistically significant(P>0.05);the relative expression of p62 in the medium and high dosage groups was improved compared with that in the model group,and the difference was statistically significant(P<0.05).Conclusions1.Qixiong formula can improve the sperm activity rate of rats with asthenospermia caused by ORN,improve the histopathological changes of epididymis,and has no significant effect on the liver and kidney functions of rats,which further the effectiveness and safety of Qixiong recipe in the treatment of asthenospermia from the animal level.2.Qixiong formula can up regulate the phosphorylation level of PI3K,Akt and mTOR protein,down regulate the expression of LC3-II/I and beclin-1 protein,up regulate the expression of p62 protein and inhibit the excessive autophagy of epididymal tissue in asthenospermia model rats,suggesting that Qixiong recipe can improve the sperm activity rate of asthenospermia model rats by activating PI3K/Akt/mTOR signal pathway and inhibiting the excessive autophagy of epididymal tissue. |
