| OBJECTIVE:In recent years,the incidence of male infertility has increased significantly,and oligospermia is one of the important causes of male infertility.Modern medicine has not fully understood the pathogenesis of oligozoospermia,and there is no specific drug treatment in clinic..The therapeutic role of traditional Chinese medicine in oligospermia is gradually recognized.TCM treatment of oligospermia is expected to become a safer and more effective treatment.Prof.Li Haisong has a unique experience in clinical treatment of weak sperm,and his curative effect is satisfactory.He uses the software of Chinese Medicine Heritage Supporting Platform System(V2.5)to analyze Professor Li Haisong's?medication rules and academic thoughts on the treatment of oligozoospermia,and based on animal experiments.The apoptotic pathway explores the molecular mechanism of its core prescription for the treatment of oligospermia and azoospermia.Method:Data Mining:Screening and collecting cases of oligospermia and azoospermia patients treated by Professor Li Haisong from September 1st,2017 to March 30th,2018 in the special needs clinic of Dongzhimen Hospital of Beijing University of Chinese Medicine,establishing a database and recording the medical prescriptions.In the the traditional Chinese medicine inheritance support system(V2.5),statistical analysis of the basic information such as the frequency of use of drugs in prescriptions,four flavors and five flavors,and returning to the classics;using unsupervised data mining methods such as association rule analysis and complex system entropy clustering,mining The association rules and prescription rules between drugs were analyzed by Professor Li Haisong's experience in treating oligospermia and azoospermia.Experimental study 1:50 SPF SD rats were randomly divided into blank control group,model control group,Chinese medicine intervention group and positive control group.Rats were treated with tripterygium glycosides(40 mg/kg)to make rats less weak.After the disease model,the blank control group and the model control group were treated with deionized water(10ml/kg).The Chinese medicine intervention group used Guangyu Yulin Decoction granule suspension(10ml/kg,3g/kg),and the positive control group was left.An aqueous solution of carnitine(10 ml/kg,0.3 mg/kg)was intragastrically administered.After four weeks of intervention,the rats were examined for general condition,body mass,organ coefficient and semen quality.Western blot and Real-Time PCR were used to detect the expression of GDNF,Ret,PI3K,Akt,Bcl-2,Bax and their mRNA.Experiment 2:30 SPF SD rats were randomly divided into blank control group,inhibitor group and Chinese medicine+inhibitor group.Blank control group:5 ml/kg of DMSO solution was injected through the tail vein from the start of the experiment,8 times per week for 8 weeks.Inhibitor group:LY294002 5 prepared by tail vein injection of DMSO from the start of the experiment.Ml/kg(0.1mg/ml),1 time/week for 8 weeks;Chinese medicine+inhibitor group:LY294002 5 ml/kg(0.1mg/ml)prepared from tail vein injection of DMSO from the start of the experiment,1 time/week,for 8 consecutive weeks,from the fifth week from 9:00 every day to the Chinese medicine granules aqueous suspension(10ml/kg,3g/kg)by intragastric intervention.Eight weeks later,the rats were examined for general condition,body mass,organ coefficient and semen quality.Western blot and Real-Time PCR were used to detect the expression of GDNF,Ret,PI3K,Akt,Bcl-2,Bax and their mRNA.Result:Data mining:A total of 398 prescriptions were included,including a total of 111 Chinese medicines,with a total frequency of 8776 times.Based on the four properties and five tastes theory in TCM,medicinal properties are mainly flat,warm and cold.Among them,the flat drugs are the most used,the warm drugs are the second,the cold drugs are used third,and the warm drugs are almost the same.Sweetness,bitterness and pungency herbal medicine were mostly used on clinical treatment,The sourness is secondly used,and the salty and astringent drugs are used less.Most medicine went to liver,kidney and spleen channel.The more frequently used medicine are are Schisandra,Gardenia,Rehmannia glutinosa,Psyllium,Cuscuta,Astragalus,Raspberry,Angelica,Chinese Yam,Oyster,Poria,Leech,Pine Pollen,Radix Paeoniae Alba,Radix Paeoniae Alba,Radix Astragali,Malt,Codonopsis,defeated grass,licorice and so on.According to the analysis of association rules,the combination of drugs for treating the core of oligospermia and azoospermia is a combination of scorpion,Schisandra,Psyllium,Cuscuta,raspberry,Rehmannia,Angelica,Astragalus,Oyster,Chinese yam,and alfalfa,based on complex system entropy.After clustering,we obtained 7 groups of new core drug combinations,and applied unsupervised entropy hierarchical.clustering algorithm to further analyze and obtain 7 new prescriptions.Experiment 1:In the testis,epididymis tissue organ,sperm count,sperm motility,the model control group,the Chinese medicine intervention group,and the positive control group were all lower than the blank control group,which was statistically significant,and the epididymis tissue organ and sperm count were Both the Chinese medicine intervention group and the positive control group were higher than the model group,which was statistically significant.In the testis,epididymis tissue organ,sperm count,improvement of sperm percentage in forward movement,there was no significant difference between the Chinese medicine intervention group and the positive control group,which was not statistically significant.The improvement of sperm total vitality was better in the Chinese medicine intervention group than the positive control group.,has statistical significane.In terms of protein and mRNA expression,compared with the blank group,the expression of GDNF,Ret,PI3K,Akt and Bcl-2 in the model control group,the Chinese medicine intervention group and the positive control group decreased to some extent,and the Bax expression increased.There were significant differences between the group and the blank control group,and the expression of each protein and mRNA was statistically significant.The expressions of RET,PI3K,Bcl-2 and Bax in the Chinese medicine intervention group and the model control group were statistically significant.There was no significant difference between the Chinese medicine intervention group and the positive control group.Experiment 2:In the testis,epididymis tissue organ,sperm count,sperm motility,the inhibition group and the Chinese medicine+inhibitor group were lower than the blank control group,the Chinese medicine+inhibitor in the epididymis tissue organ,sperm count,sperm motility The improvement effect is statistically significant.In terms of protein and mRNA expression,there was no statistical significance between GDNF and Ret.PI3K,Akt,Bcl-2,Bax expression,inhibition group and Chinese medicine+inhibitor group were lo-wer than blank control group,Chinese medicine+inhibitor There was no statistical significance between the group and the inhibition group.Conclusion:1.Professor Li Haisong's commonly used Herbal medicine for Treating oligospermia and azoospermia include Schisandra,Gardenia,Rehmannia glutinosa,Psyllium,Cuscuta,Astragalus,Raspberry,Angelica,Yam,Oyster,and Poria,among which there are more seed drugs.All core medical combinations are medlar,schisandra,psyllium,dodder,raspberry,rehmannia,angelica,scutellaria,oyster,yam and ten-flavored drugs,which also happens to be the basis of Professor Li Haisong's clinical treatment of oligozoospermia.The composition of Yu Yulin Tang.2.Professor Li Haisong's academic thoughts and medication experience in treating oligospermia and azoospermia:(1)The fundamental pathogenesis of oligospermia and azoospermia is disharmony of yin and yang,and pathological changes are based on kidney deficiency.Most of them are combined with damp-heat,blood stasis and phlegm-damp.(2)The disease of oligospermia and azoospermia was located in kidney and is closely related to the liver and spleen.(3)Teacher Li Haisong used the method Based on"nourish the kidney and strengthen the essence,and slightly adjust Yin and Yang in the treatment of oligospermia and azoospermia.Emphasized the combination of the character of medicine and paid attention to the combination of reinforcement and elimination,in order to achieve the status of "Yin and Yang in equilibrium"(4)Li Haisong's clinical treatment of oligospermia and azoospermia is based on the combination of disease differentiation and syndrome differentiation,supplemented with medicine that could tonify liver and spleen,promote Qi to activate blood and cleat heat and promote diuresis based on the basic prescription "Guangsi Yulin Decoction.3.The core prescription "Guangsi Yulin Decoction" of Professor Li Haisong could effectively improved the general state,organ coefficient and sperm quality of rats with oligospermia and azoospermia which caused by Tripterygium glycosides and inhibitor LY294002.4.The molecular mechanism of Guangsi Yulin Decoction in the treatment of oligospermia and azoospermia is closely related to the apoptosis of GDNF/Ret-PI3K/Akt-Bcl-2/Bax cells.The mechanism might be inhibiting expression of the Bax protein through promoting expression of GDNF,Ret,PI3K,Akt,Bcl.-2 protein,thereby inhibiting the apoptosis of spermatogenic cells in rats with oligospermia and azoospermia,and the mechanism of multi-target multi-channel may exist in Guangsi Yulin Decoction. |
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