| Varicocele(VC)is a common and frequently-occurring disease in andrology clinic,which is closely related to male infertility.VC can cause the quality of male semen to decline,leading to oligozoospermia and asthenospermia.Modern medicine has no specific drug for the treatment of oligozoospermia and asthenospermia caused by VC.How to improve the quality of semen has been a difficult problem in clinical treatment.The wide application of traditional Chinese medicine in clinic has greatly enriched the treatment methods of andrology diseases,expanded the treatment ideas,and has certain advantages in impro ving the quality of semen.To carry out data mining on the experience of famous old Chinese medicine in the treatment of oligozoospermia and asthenospermia and study the mechanism of Chinese medicine in improving semen quality can provide clinical reference and experimental support for the treatment of male infertility.Objective:1.Summarize Professor Li Haisong’s medication rules in the treatment of VC oligozoospermia and asthenospermia(kidney deficiency and blood stasis),excavate the coreprescription of Professor Li Haisong in the treatment of VC oligozoospermia and asthenospermia(kidney deficiency and blood stasis),explore Professor Li Haisong’s understanding of the basic pathogenesis and treatment principles of VC oligozoospermia and asthenospermia(kidney deficiency and blood stasis),and explore the way to research and inherit the Chinese medical experience with data mining methods.2.Establish the rat model of VC oligozoospermia and asthenospermia,analyze the effect of Professor Li Haisong’s core prescription for the treatment of this disease,Guangsi Tongqu Decoction,on the spermatogenesis function of the rat model of VC oligozoospermia and asthenospermia,and study its possible mechanism,providing experimental support for the clinical application of traditional Chinese medicine.Methods:Clinical study:Using the method of data mining,we collected Professor Li Haisong’s clinical Chinese medicine prescriptions for the treatment of VC oligozoospermia and asthenospermia(kidney deficiency and blood stasis),standardized the prescription drugs,and established Professor Li Haisong’s clinical Chinese medicine prescription database for the treatment of VC oligozoospermia and asthenospermia(kidney deficiency and blood stasis).The"ancient and modern medical record cloud platform" software provided by the Institute of Traditional Chinese Medicine of the Chinese Academy of Traditional Chinese Medicine is used to analyze the drug frequency,drug attribute,drug efficacy,drug association,drug clustering and complex network of prescriptions,summarize the drug use rules,extract core prescriptions,analyze the combination of prescriptions and drugs.We also conducted interviews with experts,combining quantitative research of data mining with qualitative research of expert interviews,to deeply discuss Professor Li Haisong’s understanding of the pathogenesis and treatment principles of VC induced oligoasthenospermia(kidney deficiency and blood stasis syndrome),summarize its medication rules for the treatment of this disease,extract its core prescription for the treatment of this disease,and analyze the combination of prescriptions and drugs.Experimental study:Methods of animal experiments were used,including:1.To explore the rat model of VC oligoasthenospermia:36 SD rats were randomly divided into 6 groups,with 6 rats in each group,respectively.The surgical group included a 2-week group(2-week OG),a 4-week group(4-week OG),and an 8-week group(8-week OG).The sham operation group included the 2-week sham operation group(2-week SOG),the 4-week sham operation group(4-week SOG)and the 8-week sham operation group(8-week SOG).The modified VC modeling method was used in the operation group,and the left renal vein was separated but not ligated in the sham operation group.At 2,4 and 8 weeks after the operation,samples were taken from the operation group and the sham operation group,the diameter of the left spermatic vein was measured,the pathological changes of testicular tissue were observed,and the quality of semen was measured.2.To study the effect of Guangsi Tongqu Decoction on the spermatogenesis function of VC oligoasthenospermia rat model and its mechanism:30 SD rats were randomly divided into three groups:sham operation group(Group J),drinking water group(Group Y)and Chinese medicine group(Group Z).In group J,the left renal vein was separated but not ligated.In group Y and group Z,the rat model of VC oligoasthenospermia was established by modified VC modeling method.The drug was administered 8 weeks after operation.In group J and group Y,deionized water(1ml/100g/d)was administered daily.In group Z,Guangsi Tongqu Granule suspension was administered daily(1 ml/100g/d).The gastric perfusion period was 48 days.The weight changes of rats in each group were recorded.After gavage,the diameter of the left spermatic vein was measured,and the wet weight of the left testis,organ index,semen quality,serum sex hormone and the levels of reactive oxygen species(ROS),malondialdehyde(MDA),superoxide dismutase(SOD),catalase(CAT)in testicular tissue were measured.Western blot technique was used to detect the protein expression of phosphoinositol 3 kinase(PI3K),protein kinase B(Akt),phosphorylated protein kinase B(p-Akt),forkhesd-box O class 1(FoxO1),factor associated suicide(Fas)and factor associated suicide ligand(FasL)in testicular tissue.The mRNA expression of PI3K,Akt,FoxO1,Fas and FasL in testicular tissue was detected by Real-time PCR.Results:Clinical study:A total of 224 prescriptions and 103 traditional Chinese medicines were included in this study.According to the statistical analysis of traditional Chinese medicine,Professor Li Haisong’s high-frequency drugs for the treatment of VC oligozoospermia and asthenospermia(kidney deficiency and blood stasis)include 20 kinds of drugs,such as dodder,wolfberry,astragalus,raspberry,plantain,etc.,which are more flat and warm in nature,and have more sweet,pungent and bitter in taste,and belong to more liver,kidney and spleen meridians.The efficacy classification is mainly based on drugs for tonifying deficiency and activating blood circulation and removing stasis.Correlation analysis showed that the commonly used drug pairs were "Cuscuta chinensis-wolfberry","raspberry-astragalus","astragalus-plantain","raspberry-plantain","schisandra chinensis-astragalus","schisandra chinensis-raspberry",etc.The drugs can be divided into three categories by cluster analysis.The first category has the effect of tonifying the kidney and replenishing essence,the second category has the effect of activating blood and removing stasis,and the third category has the effect of tonifying the kidney and spleen,activating blood and removing stasis.The complex network analysis obtained 11 core drug combinations,including dodder,wolfberry,plantain,raspberry,schisandra chinensis,angelica,rehmannia,astragalus,yam,achyranthes bidentata,and hot leech.After being confirmed by Professor Li Haisong,the core prescription of Professor Li Haisong for the treatment of VC induced oligozoospermia(kidney deficiency and blood stasis),Guangsi Tongqu Decoction,was extracted.Professor Li Haisong believes that kidney deficiency and blood stasis are one of the main pathogenesis of VC induced oligoasthenozoospermia,and blood stasis is a key pathological factor of VC.Kidney deficiency and oligoasthenozoospermia are closely related,and kidney deficiency and blood stasis interact,ultimately leading to this disease.The principle of tonifying the kidney and promoting blood circulation is the main treatment method for kidney deficiency and blood stasis syndrome of this disease.Experimental study:1.The rat model of VC oligoasthenospermia:compared with the corresponding 2-week SOG group,4-week SOG group and 8-week SOG group,the left spermatic vein of the 2-week OG group,4-week OG group and 8-week OG group were significantly varicose(P<0.001),the histological morphology of the testis was damaged to some extent,and the PR+NP decreased significantly(P<0.001).Although the sperm concentration of 2-week OG group decreased,the difference was not statistically significant(P>0.05).The sperm concentration of 4-week OG group and 8-week OG group decreased significantly(P<0.01).Compared with the 2-week OG group,the left spermatic vein of the 4week OG group was significantly more varicose(P<0.01),the testicular tissue morphology was more severely damaged,and the sperm concentration and PR+NP were significantly decreased(P<0.01).Compared with the 4-week OG group,the left spermatic vein of the 8week OG group was significantly varicose(P<0.05),the histological morphology of the testis was more severely damaged,and the sperm concentration and PR+NP were significantly decreased(P<0.05 or P<0.01).2.Effect of Guangsi Tongqu Decoction on spermatogenesis function of VC oligoasthenospermia rat model and its mechanism:Compared with group J,the wet weight of testis in group Y decreased significantly(P<0.001),the organ coefficient decreased significantly(P<0.001),the diameter of left spermatic vein increased significantly(P<0.001),the histological morphology of testis was damaged,the sperm concentration decreased significantly(P<0.01),and the PR+NP decreased significantly(P<0.001).The serum testosterone(T)level decreased significantly(P<0.001),follicle-stimulating hormone(FSH),luteinizing hormone(LH)level increased significantly(P<0.001),ROS and MDA levels in testicular tissue increased significantly(P<0.001),SOD and CAT levels decreased significantly(P<0.001).The expression of PI3K protein and mRNA decreased significantly(P<0.05 or P<0.001),the expression of Akt protein and mRNA decreased significantly(P<0.01 or P<0.001),the expression of p-AKT protein decreased significantly(P<0.01),the expression of FoxO1 protein and mRNA increased significantly(P<0.05 or P<0.001),the expression of Fas protein and mRNA increased significantly(P<0.01 or P<0.001),and the expression of FasL protein and mRNA increased significantly(P<0.01 or P<0.001).Compared with group Y,the wet weight of testis in group Z increased significantly(P<0.05),the organ coefficient also increased,but there was no statistical significance(P>0.05),the diameter of left spermatic vein did not change significantly(P>0.05),the histological morphology of testis basically returned to normal,sperm concentration increased significantly(P<0.01),and PR+NP increased significantly(P<0.001).The serum T level increased significantly(P<0.05),FSH and LH levels decreased significantly(P<0.01 or P<0.05),ROS and MDA levels decreased significantly(P<0.001),and SOD and CAT levels increased significantly(P<0.01 or P<0.001).The expression of PI3K protein and mRNA increased significantly(P<0.01 or P<0.001),the expression of Akt protein and mRNA increased significantly(P<0.001),the expression of p-AKT protein increased significantly(P<0.01),the expression of FoxO1 protein and mRNA decreased significantly(P<0.05 or P<0.001),the expression of Fas protein and mRNA decreased significantly(P<0.05 or P<0.01),and the expression of FasL protein and mRNA decreased significantly(P<0.01 or P<0.001).Conclusions:1.Professor Li Haisong believes that the main pathogenesis of VC induced oligoasthenozoospermia is kidney deficiency and blood stasis.The treatment focuses on tonifying the kidney and promoting blood circulation,with Guangsi Tongqu Tang as the core prescription.2.VC can lead to spermatogenesis dysfunction in rats.The model of VC oligozoospermia and asthenospermia rat model prepared by the improved VC modeling method with 8 weeks as the modeling cycle is the most ideal.3.Guangsi Tongqu Decoction can improve the testicular wet weight,sexual hormone levels,semen quality,and the morphological structure of testicular tissue in the rat model of VC induced oligoasthenospermia,and can also regulate the levels of oxidation and oxidation resistance,restore the balance between oxidation and oxidation resistance systems,and reduce the damage of oxidative stress to the testis4.The mechanism of Guangsi Tongqu decoction in improving the spermatogenesis function of VC oligoasthenospermia rat model may be related to the regulation of PI3K/Akt/FoxO1 and Fas/FasL signaling pathway.To sum up,this study explored Professor Li Haisong’s understanding of the pathogenesis and treatment principles of VC oligoasthenospermia(kidney deficiency and blood stasis),summarized Professor Li Haisong’s medication rules and core prescriptions for the treatment of VC oligoasthenospermia(kidney deficiency and blood stasis),and provided experience for the current treatment of VC oligoasthenospermia.The rat model of VC oligozoospermia and asthenospermia was established,and the effect of Guangsi Tongqu Decoction on the spermatogenic function of VC induced oligozoospermia and asthenospermia rats and its possible mechanism were analyzed,which provided experimental support for the treatment of VC oligozoospermia and asthenospermia with traditional Chinese medicine. |
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