The Alleviating Effect And Its Mechanism Of Schisandrin B On Mouse Orchitis

With the aging of the population,infertility has gradually become a prominent social problem,and the increase in male fertility pressure has also caused widespread concern in society.Similarly,the breeding ability of various rare and endangered animals,wild animals and breeding males is also worthy of attention.Orchitis is one of the main causes of male infertility.It often occurs in farms,and the presence of this inflammation can seriously affect the reproductive ability of male animals;and in severe cases,it will cause azoospermia and even be life-threatening.At present,antibiotics are still the first choice of drugs to treat orchitis but high levels of antibiotics can lead to the emergence of resistant strains and create problems with antibiotic residues.Therefore,it is particularly important to research and develop new highly effective,drug-free and residue-free drugs.A large number of clinical practices have proven that,compared with antibiotics,herbal medicines have the advantages of low toxicity,no drug resistance and residual problems,and have a beneficial role in the prevention and treatment of inflammation.Schisandra chinensis is the dominant herbal medicine in Northeast China,which has many effects such as calming,anti-inflammatory and antioxidant etc.The main active ingredients of Schisandra chinensis are the lignans,such as Schisandrin,Schisandrin B(SchB),etc.Our previous studies have found that SchB has a positive effect on the adult neurogenesis,but its effect and mechanism on testicular inflammation remain unclear.Based on the research background,we used lipopolysaccharide(LPS)induction to simulate bacterial infection in mice and mouse Sertoli cells(mSCs),prepare orchitis model in vivo and in vitro,and investigated the effects and its mechanisms of the natural compound SchB on ochitis,aiming to provide new ideas and lay a theoretical foundation for the prevention of orchitis in domestic animals.In the in vivo experiments,C57BL/6 mice pretreated with SchB at different concentrations were injected intraperitoneally with LPS for 3 days and sacrificed 12 hours later to take both testies for follow-up experiments.The results showed that the seminiferous epithelium was seriously damaged and no sperm was abserved in the tubules of LPS group after hematoxylin-eosin(HE)staining.In addition,LPS stimulation significantly increased the expression of inflammatory mediators,including interleukin-6(IL-6),interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),cyclooxygenase 2(COX-2)and nitric oxide synthase(iNOS),while SchB pretreatment significantly decreased these changes.The above results suggest that SchB can alleviate LPS-induced orchitis in mice and attenuate LPS-induced inflammatory mediators in mouse testes.The testicular Sertoli cells(SCs)play important role in spermatogenesis.In this study,LPS stimulation of mouse SCs(mSCs)was used to prepare the in vitro model of ochitis,for examining the expressions of IL-6,IL-1β,TNF-α,COX-2 and iNOS mRNA.The mRNA levels of these cytokines were significantly reduced after SchB pretreatment.To assess the possibility that NF-κB and MAPK signaling pathways are involved in the anti-inflammatory effects of SchB in LPS-stimulated mSCs,we used the above in vitro model and collected the cells for western blotting.The results showed that LPS activated the NF-κB and MAPK signaling pathways,while they were inhibited after SchB pretreatment.These results imply that SchB can alleviate LPS-induced orchitis in vitro,and attenuate the inflammatory mediators of LPS-induction by suppressing NF-κB/MAPK signaling pathway.Studies have shown that inflammation causes cell apoptosis.In this study,the expression of apoptotic marker Bax and Caspase-3 protein was significantly increased after LPS stimulation,and the expression of anti-apoptotic marker Bcl2 was significantly decreased.Tunel staining showed that the apoptotic mSCs increased significantly in LPS group,while SchB pretreatment could improve the above phenomena.To further understand the underlying mechanism,the cells were incubated with the activator Anisomycin(5 μM)of the key molecule JNK in MAPK pathway for one hour prior to SchB and LPS treatment.After Anisomycin pretreatment,apoptosis of mSCs is greatly reactivated.Further Anisomycin activation again increased the number of apoptotic cells.These data suggest that the MAPK(JNK)signaling pathway is involved in SchB inhibition of m SC apoptosis.Taken together,our results suggest that SchB reduces the damage to testicular tissue and the production of pro-inflammatory mediators in testes.These protective effects are mediated by inhibiting the activation of NF-κB and MAPK signaling pathways.In addition,SchB attenuates LPS-induced apoptosis,which was initiated at least in part by inhibiting the MAPK(JNK)signaling pathway.Overall,our results suggest that SchB has a promising therapeutic effect in cell and mouse models of LPSinduced orchitis,and can be used as a promising precursor compound for the clinical prevention of orchitis.