Effect Of Shungan Bushen Yulin Decoction On VDAC2 And CAMP/PKA Pathway Expression In Rats With Hypoasthenospermia And Its Mechanism

ObjectiveTheoretically elucidate the TCM pathogenesis of"stagnation of liver and deficiency of kidney"of oligospermia,and to observe the clinical effect of shugan bushen yulin decoction on oligospermia.E-stablishing a adenine lavage combined liver kidney empty type of chronic bondage stress caused by less weak sperm disease rat model.To investigate the therapeutic effect of Sooging the Liver and Bending the Kidney Yulin Decoction on oligospermia of liver stagnation and kidney deficiency rat model from the perspective of VDAC2 and cAMP/PKA pathway expression and the mechanism of action,to provide ideas for clinical treatment.Methods1.Theoretical research:To sort out the history of Chinese medicine of oligospermia,this paper discusses the TCM pathogenesis of oligospermia and clarifies the theoretical mechanism of treating oligospermia by shugan bushen.2.Clinical study:80 male patients who were clinically consistent with the diagnosis of oligospermia of liver stagnation and kidney deficiency type were randomly divided into two groups,among which 40 patients in the treatment group were treated with shugan bushen yulin decoction,and 40 patients in the control group were treated with Qilin Pill.Both groups were treated for 3 months as a course of treatment.The changes of semen volume,sperm concentration and sperm motility(PR grade)were observed in the two groups before and after treatment.3.Experimental study:Sixty-five male SD rats were randomly divided into blank group,model group,western medicine group,high dose Chinese medicine group and low dose Chinese medicine group,with 13 rats in each group.Adenine intragastric administration and chronic binding stress stimulation were used to establish the rat model of oligoasthenospermia with liver stagnation and kidney deficiency.The process took 21 days.In the first 3 weeks,rats in model group,western medicine group,high dose Chinese medicine group and low dose Chinese medicine group were gavaged with adenine(1m L/100g/d)according to body weight for 14 consecutive days.Meanwhile,rats were bound with a restraining tank every day(3hours/time,twice/day).The blank group was fed normally.On the last day of week 3,3 rats from each group were randomly selected for behavioral analysis,serum cAMP and c GMP,sperm quality was detected,and testis pathological sections were observed under light microscope to determine whether the model was successful.From the 22nd day,the traditional Chinese medicine group was given shugan bushen yulin decoction by gavage.According to the ratio of the reduced dose of rat surface area to human body,the gavage doses were 32.4g/Kg/d and 8.1g/Kg/d,once a day,respectively.Weight was weighed every other day during administra-tion,and the dose was adjusted according to body weight for 28 consecutive days.Rats in blank group and model group were given equal amount of deionized water by gavage for 28 consecutive days.During the modeling process,the fur,food intake,activity state and other general conditions of each group were observed every day.Serum cAMP,c GMP and sex hormone testosterone(T)content were detected by ELISA and histological changes of testis were observed by light microscope with HE staining after model formation.By detecting VDAC2methylation;The expressions of VDAC2,PKA and AKAP4 were detected by immunofluorescence assay,fluorescence quantitative PCR and Western BOLT.The concentrations of Ca²⁺and ATPase were determined by kit method;The mitochondrial membrane potential was detected by flow cytometry.ResultTheoretical research:Liver stagnation and kidney deficiency is one of the important basic pathogenesis of oligosthenospermia.Liver stagnation and kidney deficiency affect each other,and deficiency and deficiency are mixed.Shugan bushen is an important treatment method for oligosthenospermiaClinical study:The clinical total effective rate of the control group was 55%,compared with that of the treatment group was80%,the difference was statistically significant(P<0.05).There was no difference in semen volume between control group and treatment group before and after treatment(P>0.05);Compared with the control group,the sperm density and motility in the treatment group were significantly increased,and the differences were statistically significant(P<0.01).Experimental study:Compared with blank group,diet intake,body weight,reproductive weight,reproductive organ weight,cAMP,cAMP/c GMP ratio,testosterone,sperm density and activity rate,m RNA and protein expressions of VDAC2,AKAP4 and PKA,Ca²⁺and ATPase,mitochondrial membrane potential in model group were significantly decreased(P<0.01 or 0.01<P<0.05;c GMP and VDAC2 methylation were significantly increased(P<0.01).Compared with model group,food intake,body weight and genital weight of western medicine group,Chinese medicine high-dose group and Chinese medicine low-dose group decreased cAMP,cAMP/c GMP ratio,testosterone,sperm density and activity rate,m RNA and protein expression of VDAC2,AKAP4 and PKA,Ca²⁺and ATPase,and mitochondrial membrane potential increased(P<0.01 or 0.01<P<0.05);c GMP and VDAC2 methylation decreased(P<0.01).Compared with the western medicine group,AKAP4(0.01<P<0.05),the others had no difference;There was significant difference in the low dose group(P<0.01).Conclusion1.The pathogenesis of oligospermia is a mixture of deficiency and deficiency,mainly manifested as stagnation of liver and deficiency of kidney.The treatment of shugan bushen is an effective for oligospermia.2.Shugan bushen yulin decoction has a significant effect on the treatment of oligospermia.3.Compound model was successfully established by adenine gavage combined with chronic binding stress stimulation.4.Shugan bushen yulin decoction can significantly improve sperm quality and general symptoms of model rats.5.Shugan bushen yulin decoction can regulate the secretion of sex hormone testosterone by regulating the HPG axis,and increase the quality of testis and epididymis to improve the sperm count of model rats.6.Shugan bushen yulin decoction can enhance the expression of VDAC2 m RNA and protein by inhibiting VDAC2 methylation,thus increasing the concentration of intracytoplasmic Ca²⁺,and then changing the mitochondrial membrane potential and ATP concentration to enhance sperm motivity.7.Shugan bushen yulin decoction can improve the activity of PKA by regulating the cAMP/PKA pathway,and to a certain extent increase the expression of AKAP4,thus enhancing the ATP output of mitochondria and enhancing the motility of sperm.8.In addition to tyrosine phosphorylation of cAMP/PKA,glycolysis of Akap4 may be another important pathway for sperm capacization.