Study On Regulation Of Nerve Growth Factor On Sertoli Cells Proliferation And Inflammatory Injury In Dairy Goats

In animal husbandry,it is important to maintain stable spermatogenesis for the reproductive ability of breeding males.Spermatogenesis requires close cooperation between germ cells and a variety of somatic cells,among which Sertoli cells(SCs)can provide a stable microenvironment for spermatogenesis.The blood-testosterone barrier formed by conjunctions between SCs and germ cells can ensure the orderly progress of spermatogenesis.Foreign toxic substances can cause imbalance of cell homeostasis,produce inflammatory response,damage of SCs junctions and affect spermatogenesis,which may lead to male sterility.Nerve growth factor(NGF)is secreted by SCs in testis and acts on SCs through SCs surface receptor Trk A.It plays a crucial role in diseases of male reproductive system and inflammation-related diseases.However,it is not clear how NGF regulates the function of dairy goat SCs.Therefore,in this study,the primary SCs of dairy goats were firstly extracted,identified and treated with NGF to detect the positive rate of Ed U,intracellular ATP content and apoptosis-related gene m RNA levels,and to explore the effects of NGF on the growth of dairy goats SCs.Secondly,the SCs was treated with LPS to detect the m RNA levels of inflammatory genes.The effects of LPS on the expression of junctions in SCs were detected by ultrastructural observation,immunofluorescence staining and Western blot.Finally,after LPS treatment,NGF was co-treated with LPS,and Lactate dehydrogenase(LDH)was used to detect cytotoxicity and explore the effect of NGF on LPS-induced conjunctin damage.The effects of NGF and LPS on cell barrier integrity were evaluated by transmission electron microscopy and transepithelial electrical resistance(TEER).Western blot was used to detect the activation of PI3K/AKT/NFκB signal,so as to reveal the regulation and mechanism of NGF on the proliferation and inflammatory injury of dairy goats.The main results of this study are as follows:1.The testis of dairy goats aged 2～3 months were collected and the primary SCs were obtained by mechanical separation,enzyme digestion and differential adhesion.Morphological identification by scanning electron microscopy showed that the SCs was irregular and the nuclei were oval,and the long axis of the nucleus was parallel to the long axis of the cell,which was consistent with the morphological characteristics of the SCs.The expression of SCs specific protein SOX9 and WT1 were detected by immunofluorescence,and the cell purity was 96.1% ± 0.8% and 95.1%±1.2% by flow cytometry.SCs were treated with 25,50,100,200 ng/m L NGF for 12,24,36,48 h,CCK-8cell proliferation rate was detected,it was found that 200 ng/m L NGF treatment for 24 h,cell proliferation rate was significantly higher than the control group(P < 0.01);In addition,Ed U positive rate and ATP content in 200 ng/m L group were significantly higher than those in control group(P < 0.01);q PCR showed that the m RNA level of pro-apoptotic gene Caspase3 was significantly lower than that of the control group(P < 0.01),but Bax m RNA level was significantly lower than that of control group(P < 0.05),the m RNA level of anti-apoptotic gene Bcl-2 was significantly higher than that of control group(P < 0.01).These results indicate that NGF can promote the proliferation and inhibit the apoptosis of dairy goat SCs.2.In order to investigate the effect of LPS on conjunctin,SCs were treated with 5,10,25,50,100,250,500,1000 and 2000 ng/m L LPS for 24 h,respectively,and the cell viability inhibition rate was determined by CCK-8.The results showed that the cell viability inhibition rate of 1000 ng/m L LPS group was 50%.The results of q PCR showed that the m RNA levels of pro-inflammatory factors IL-1β and TNF-α were significantly higher than those of control group(P < 0.01),the m RNA level of anti-inflammatory factor IL-10 was significantly lower than that of control group(P < 0.01);The m RNA levels of pro-apoptotic genes Caspase3 and Bax were significantly higher than those of control group(P < 0.01),the m RNA level of anti-apoptotic gene Bcl-2 was significantly lower than that of control group(P < 0.01);In addition,scanning electron microscopy showed that LPS treatment could cause damage of filamentous pseudopodia.Immunofluorescence and Western blot showed that the expression levels of Occludin,CX-43 and β-Cantenin were significantly lower than those of the control group(P < 0.01).These results showed that the SCs underwent inflammatory response and induced junctions damage after treatment with1000 ng/m L LPS for 24 h.3.In order to investigate the effect of NGF on LPS-induced cell damage,cells were treated with 1000 ng/m L LPS for 6,12 and 24 h,and cultured with 1000 ng/m L LPS and200 ng/m L NGF for 24 h from 24 h to 48 h,respectively(LPS+NGF).LDH test showed that NGF could block the cytotoxicity of LPS time-dependent mode.The expression levels of Occludin,CX-43 and β-cantenin in LPS+NGF group were significantly higher than those in LPS+M group(P < 0.01);TEER detection showed that the TEER value in LPS+NGF group was significantly higher than that in LPS+M group(P < 0.01);Western blot showed that NGF inhibited LPS-activated PI3K/AKT/NFκB signaling.The results of q PCR showed that m RNA levels of Caspase3,Bax,IL-1β and TNF-α in LPS+NGF group were significantly lower than those in LPS+M group(P < 0.01),the m RNA levels of Bcl-2and IL-10 in LPS+NGF group were significantly higher than those in LPS+M group(P <0.01).This study showed that NGF can resist LPS-induced inflammation and cell damage through the PI3K/AKT/NFκB signaling pathway.In conclusion,NGF can promote the proliferation,inhibit apoptosis and increase intracellular ATP content of dairy goat SCs.1000 ng/m L LPS can induce cell inflammation and decrease junctions expression,while NGF can resist LPS-induced inflammation and cell damage through PI3K/AKT/NFκB signaling pathway.Therefore,this study clarified the protective effect of NGF on dairy goats SCs,providing a reference for the systematic study of male reproductive mechanism in large mammals such as domestic animals,and laying a foundation for the treatment of testicular diseases in mammals.