Based On Bcl-2/Bax Pathway To Explore The Mechanism Of Testudinis Carapacis Et Lastri Colla In The Treatment Of Infertility With Oligoasthenospermia Due To Deficiency Of Kidney Yin

Objective:According to the epidemiological survey,the incidence of male oligoasthenospermia infertility is increasing day by day,but the treatment of this disease is still very limited,the therapeutic effect is not satisfactory,and assisted reproductive technology costs a lot,drug treatment lacks specific drugs.Chinese medicine tortoisesha gum taste salty,sweet,cool.Gui liver,kidney and heart meridian are solid glue made of Chinese traditional medicine tortoisesha after boiling and concentrating.It has the function of nourishing Yin and kidney,nourishing blood and stopping bleeding.It mainly treats Yin deficiency,hot flushes,bone steaming night sweats,waist and knees sour and soft,blood deficiency and verticality,breaking and leaking band and so on.The purpose of this study was to investigate TESTUDINIS CARAPACIS ET LASTRI COLLA of kidney Yin deficiency type less weak sperm sperm,sperm vitality of rats,serum testosterone levels,mitochondrial function,the effect of cell apoptosis index and corresponding protein expression of intervention,aimed at men less weak sperm fertility medications for new treatment options.Methods:1.Animal grouping:Male Sprague-Dawley（SD）rats 200±20g,10 weeks old.The rats were randomly divided into 7 groups:normal group,model group,tortoise shell low-dose group,tortoise shell medium-dose group,tortoise shell high-dose group,positive control group,tortoise shell glue+L-carnitine group,15 rats in each group.All animal procedures were approved by the Animal Welfare Committee of Hunan University of Traditional Chinese Medicine.2.Preparation of animal model:The preparation of the rat model of oligoasthenospermia with deficiency of kidney yin is induced by levothyroxine.The daily dose of levothyroxine was 45ug·kg^-1·d^-1by gavage for 30 consecutive days,during which the condition of the model group was observed.3.Drug intervention:After the model is successfully prepared,the drug will be administered on the first day after the animal model is successfully established.The normal group and the model group are given normal saline by gavage,and the tortoise shell gum low,medium and high dose groups are given 0.5g respectively/kg/d,1g/kg/d,2g/kg/d tortoise shell glue,the positive control group was given 8ml/kg/d of L-carnitine,the tortoise shell glue+L-carnitine group was given 2g/kg/d Tortoise shell glue+8ml/kg/d Levocarnitine for 30 days.During this experiment,the rats were weighed once a week,and the dosage of each rat was adjusted according to the weighing results.After the drug intervention process was over,the rats were sacrificed with pentobarbital sodium.4.Test the following indicators:（1）Use liquid chromatography-mass spectrometry to determine the amino acid content of turtle shell gum;（2）Detect the quality of rat testis tissue in each group;（3）Use computer-aided analysis method（CASA）to analyze the semen quality of rats in each group;（4）Use flow cytometry to detect the total apoptosis rate of rat spermatogenic cells in each group;（5）Use flow cytometry to detect the function of the mitochondrial permeability transition pore（m PTP）of rat spermatogenic cells in each group;（6）Use enzyme-linked immunosorbent assay（ELISA）to detect serum testosterone（T）levels of rats in each group;（7）Western Blot was used to detect the anti-apoptotic protein（Bcl-2）,pro-apoptotic protein（Bax）,（cleaved-caspase3）and the key proteins VDAC and m PTP function of rat spermatogenic cells in each group.ANT;（8）Use enzyme-linked immunosorbent assay（ELISA）to detect the mitochondrial respiratory chain complexes of rat spermatogenic cells（respectively:NADH-Q oxidoreductase（complex I）,succinate-Q oxidoreductase（complex）Compound II）,UQ-cytochrome C oxidoreductase（complex III）,cytochrome C oxidase（complex IV）,ATP synthase（complex V）)activities;（9）Use hematoxylin-eosin staining（HE）to observe the changes in the distribution of spermatogenic cells and mesenchymal cells in the testes of each group.Results:1.Amino acid content of tortoisescape gum:18 amino acids were detected in traditional Chinese medicine tortoisescape gum,and the contents of which were 18.91%alanine（ALA）,12.60%glycine（Gly）,6.83%proline（Pro）,6.52%glutamine（Glu）,5.49%hydroxyproline（Hyp）and 5.02%arginine（Arg）,among which the contents were more than 5%.2.The quality of testis in rats:compared with the normal group,the mass of testis in model group was significantly decreased（P<0.01）;Unilateral testicular mass in TCLC low-dose group,TCLC medium-dose group,TCLC high-dose group,positive control group and TCLC+L-carnitine group was significantly higher than that in model group（P<0.01）.Unilateral testis mass of TCLC high-dose group was significantly higher than that of positive control group（P<0.05）.There was no significant difference in testicular mass between TCLC high-dose group and TCLC+L-carnitine group（P>0.05）.3.Semen quality of rats:The semen parameters of rats mainly included sperm curve velocity（VCL）（μm/s）,sperm motility（%）and sperm concentration（×10⁶）.Compared with model group,the linear velocity（μm/s）,sperm motility（%）and sperm concentration（×10⁶）of medium dose and high dose tortoisesol groups were significantly improved（P<0.05）.Compared with the positive control group,sperm concentration in tortoisesol+levocarnitine group was significantly improved（P<0.05）;4.The total apoptosis rate of spermatogenic cells in rats:compared with the normal group,the total apoptosis rate of spermatogenic cells in the model group was significantly increased（P<0.01）,and the total apoptosis rate of spermatogenic cells in the middle dose group,high dose group,positive control group,and L-carnitine group was significantly decreased compared with the model group（P<0.05）.5.Rat spermatogenic cell MPTP:Compared with model group,the opening of MPTP of spermatogenic cells in medium dose group,high dose group,positive control group,and levocarnitine group was significantly improved（P<0.05）;6.Serum T level of rats:Compared with model group,serum T level of rats in medium dose group,high dose group,positive control group and L-carnitine group was significantly increased（P<0.05）;Compared with the positive control group,the serum T level in tortoise-glue+L-carnitine group was significantly increased（P<0.05）.7.Bcl-2,Bax,cleaved caspase3 and MPTP key proteins VDAC and Ant in spermatogenic cells of rats:compared with normal group,Bcl-2 in spermatogenic cells of rats of model group was significantly decreased（P<0.05）;Spermatogenic cell Bcl-2 in middle dose group,high dose group,positive control group and L-carnitine group was significantly higher than that in model group（P<0.05）.Compared with the normal group,Bax and cleaved caspase3 in spermatogenic cells of model group were significantly increased（P<0.05）.Spermatogenic cell Bcl-2 in middle dose group,high dose group,positive control group and L-carnitine group was significantly lower than that in model group（P<0.05）.Compared with the normal group,VDAC and ANT of spermatogenic cells in model group were significantly decreased（P<0.05）.VDAC and ANT of spermatogenic cells in medium dose group,high dose group,positive control group and levocarnitine group were significantly higher than those in model group（P<0.05）.8,the rat sperm cells mitochondrial respiratory chain complexesⅠ-Ⅴ:compared with normal group,model group rats born sperm cells mitochondrial respiratory chain complexesⅠ-Ⅴactivity significantly decreased（p<0.05）;TESTUDINIS CARAPACIS ET LASTRI COLLA dosage group,the tortoise shell in high dose group,positive control group,TESTUDINIS CARAPACIS ET LASTRI COLLA+l-carnitine group rats born sperm cells mitochondrial respiratory chain complexesⅠ-Ⅴactivity is significantly higher than the model group（p<0.05）;9.Changes in the distribution of spermatogenic cells and mesenchymal cells in rat testis;Compared with the normal group,the testicular tissue of rats in the model group was obviously damaged,including atrophy and destruction of spermatogenic cells,significantly reduced number of layers,significantly reduced number of sperms in the lumen,and significantly degenerated,edema and loose interstitium.Compared with model group,the above changes were significantly improved in high-dose group,positive control group and L-carnitine group.Conclusion:1.The pathogenesis of oligoasthenospermia with kidney-yin deficiency is closely related to the abnormal expression of Bcl-2,Bax,cleaved caspase3and abnormal energy metabolism.2.Chinese traditional medicine chaisei jiao can significantly improve the semen quality of rats with oligoasthenozoospermia of kidney-yin deficiency type.The main mechanism of action is as follows:（1）reduce the apoptosis rate of spermatogenic cells in rats,and contribute to the increase of sperm count;（2）Improve the function of MPTP in spermatogenic cells and improve sperm motility;（3）Improve serum T level to promote spermatogenesis;（4）Regulating the expression of Bcl-2,Bax,cleaved caspase3,effectively inhibiting the apoptosis of spermatogenic cells,promoting the proliferation of spermatogenic cells,and increasing sperm count;（5）Improve the activity of mitochondrial respiratory chain complex I-V in spermatogenic cells,which can improve sperm respiration and sperm motility.（6）Reduce the pathological damage of testicular tissue and improve the environment of sperm development.