Effect Of Diaphragma Juglandis Fructus On Asthenozoospermia Mice And Prostatitis Rats

Objective:Using the extract of Diaphragma Juglandis Fructus（DJF）as the experimental object,the active site and mechanism of its action in the treatment of asthenozoospermia mice and chronic non-bacterial prostatitic rats were explored.The results will provide a research basis for the modern development and utilization of the traditional medicine use of DJF.Methods:1.Study on the active site and mechanism of DJF in the treatment of asthenozoospermia mice.（1）Screen of the active site of DJF on asthenozoospermia mice.Asthenozoospermia mice was established by cyclophosphamide and were given different extracts of DJF by gavage for intervention,once per day for 25 consecutive days.After the last administration,the sperm quality,testosterone level and testicular histopathological changes were detected to screen the active site of DJF on asthenozoospermia mice.（2）Study on the mechanism of water extract of DJF on asthenozoospermia.The target and pathway of DJF on asthenozoospermia was predicted with network pharmacology.The water extract that had the best activity via above experiment was roughly divided by D101macroporous resin column chromatography,and then the mechanism of action was further studied.In detail,the water extact of DJF（DJF-W）was divided into three elusion sites,that is the water elution site（D101-1）,the 50%methanol elution site（D101-2）and the 100%methanol elution site（D101-3）,respectively.Then,the contents of total flavonoids,total polyphenols and total polysaccharides in each part were detected by chemical method.About mechanism study,asthenospermia mice was established as above method,then,the testosterone level in the serum of asthenospermia mice were detected by enzyme linked immunosorbent assay（ELISA）,the content of lactate dehydrogenase（LDH）and superoxide dismutase（SOD）in the serum were measured,and the malondialdehyde（MDA）and glutathione（GSH）level in testicular tissue were measured by colorimetry.Moreover,the m RNA and protein expression level of B-cell lymphoma 2（Bcl-2）and Bcl2 associated X protein（Bax）in testicular tissue were detected by RT-q PCR and western blot.2.Study on the active site of DJF and mechanism of action on chronic non-bacterial prostatitis rats.（1）Screen of effective anti-inflammatory sites of DJF:After preparation of the water extract of DJF and its D101 macroporous resin elution sites,RAW264.7 macrophage inflammation model was induced by lipopolysaccharide（LPS）.After that,the water extract of DJF and its elution sites were added for incubation.Then,the level of nitric oxide（NO）in cell supernatant was determined to screen the anti-inflammatory sites of DJF.The results showed that the water extract of DJF had the most effective anti-inflammatory action.（2）Study on the effect and mechanism of water extract of DJF on chronic non-bacterial prostatitis rats.The chronic non-bacterial prostatitis（CNP）rat model was established with carrageenan,and the rats were treated with the water extract of DJF,once per day for 10consecutive days.1 hour after the last administration,rats were weighed,and prostate tissue was taken to calculate the prostate index（PI）.Then,blood was taken,and the content of prostate specific antigen（PSA）in serum was detected by ELISA.The histopathological changes of prostate were observed by H&E staining.The contents of tumor necrosis factor-α（TNF-α）and prostaglandin E2（PGE₂）in the serum were detected by ELISA.The content of malondialdehyde（MDA）in prostate tissue was detected by colorimetric method.The gene expression levels of interleukin（IL-10）,cyclooxygenase 2（COX-2）and NF-E2 related factor2（Nrf2）in tissues were detected by RT-q PCR to explore its mechanism of action.Results:1.Study on the active site and mechanism of DJF in the treatment of asthenozoospermia mice.（1）Study on the active site of DJF on asthenozoospermia mice.All the water extract,the deionized water extract and the ethanol extract of DJF could increase the testicular weight of asthenospermia mice,improve the quality of sperm and the content of testosterone in serum,increase the number of testicular spermatid and deformed spermatocytes in the lumen.Among them,the effect of the water extract of DJF was the best.（2）Study on the mechanism of water extract of DJF on asthenozoospermia.The network pharmacology result showed that the major active ingredients of DJF are flavonoids,phenolic acids,etc.The main effects of DJF on asthenozoospermia were related to gonadal development,eproductive system development,steroid hormone response,androgen metabolism process,cell response to oxidative stress and apoptosis,etc.The experimental results showed that the content of total flavonoids in DJF-W,D101-1,D101-2 and D101-3 is48.84±0.93,19.01±4.37,87.53±2.73 and 65.37±4.92 respectively.The total polyphenols content is 17.39±0.49,12.60±1.57,26.62±0.81 and 20.89±0.57 respectively.The total polysaccharides content is 9.58±1.07,9.99±0.92,14.39±1.29 and 7.30±0.74 respectively.Compared with the model group,the water extract of DJF and its elution sites could increase the testicular weight of mice and the content of testosterone in the serum,improve the quality of sperm,and increase the number of testicular spermogenic cells and deformed sperm cells in the lumen.At the same time,the water extract of DJF and its elution sites could reduce the contents of LDH and MDA,increase the contents of SOD and GSH,enhance the expression level of anti-apoptotic Bcl-2 gene and pro-apoptotic Bax gene in asthenospermia mice,significantly enhance the expression level of anti-apoptotic Bcl-2 protein and reduce the pro-apoptotic Bax protein expression level.2.Study on the active site and mechanism of DJF in the treatment of chronic non-bacterial prostatitis rats:（1）Screen of effective anti-inflammatory components of DJF.The water extract of DJF and its elution sites could inhibit the release of NO induced by LPS,and the water extract of DJF had the best effect.（2）Study on the effect and mechanism of water extract of DJF on chronic non-bacterial prostatitis rats.The water extract of DJF could reduce the PSA level in the serum of CNP rats,alleviate prostate tissue edema and inflammatory cell infiltration,reduce the content of TNF-αand PGE₂ in the serum,and the level of MDA in tissues,reduce the gene expression level of IL-10 and COX-2 in tissues,and increase the gene expression level of Nrf2.Conclusion:1.The water extract of DJF is the most active site for the treatment of asthenospermia.The water extract of DJF and its elution sites could treat asthenospermia mice by inhibiting oxidative stress response and apoptosis.2.The water extract of DJF could treat CNP rats by inhibiting the release of inflammatory factors and anti-oxidation.Its mechanism of action was related to COX-2/PGE2 and Nrf2 pathway.3.DJF has the effect on the reproductive and urinary disease.Its traditional therapeutic effect is reasonable.