| [Purpose] To investigate the localization and expression of Ca SR in primary testicular macrophages in UPEC-induced orchitis model.[Methods] A 250-300 kg Wistar male rat was Purchased from the Hubei Provincial Center for disease Control and Prevention to establish a UPEC orchitis model to collect testes,interstitial fluid,and blood specimens.First,shake UPEC bacteria solution in LB antibiotic-free medium overnight,and check the quality of the UPEC bacteria solution with a microplate reader;resuspend the UPEC bacteria with 1 ml of sterile normal saline,configure the bacteria suspension,and inject the bacteria solution retrogradely from the vas deferens of both sides of the rat.After stitching the wound.An equal amount of saline was injected as a Control group.After 7 days,it was confirmed whether the model was successfully constructed.The experimental methods included identification of UPEC gene PAPC and colonies,testicular HE staining,and searching for UPEC bacteria in the testicles under an electron microscope.After confirming that the model was successfully constructed,fresh testis was taken to isolate primary testis macrophages,to identify the Purity of the cells,and to detect the expression of Ca SR in macrophages.[Results] Seven days after the model was established,UPEC group’s testicular homogenate clearly contained UPEC bacteria PAPC gene;and after the UPEC group testicular homogenate was coated on the Plate,a large number of UPEC bacterial colonies were generated by the naked eye the next day,while the Control Plate had Bacterial colonies grow.Under the electron microscope,UPEC bacteria were found in the testis interstitium of the UPEC infected group.macroscopically,the testes of the UPEC group were reduced in volume and reduced in mass;compared with the Control group,the forward sperm motility and total sperm count were reduced,and the sperm quality was significantly affected.In testicular tissues,the results showed that Ca SR was expressed in normal rat testis tissues,and Ca SR m RNA and Protein levels were significantly increased after infection with UPEC bacteria.The results of TM RT-PCR and WB also showed that Ca SR was highly expressed in TM of UPEC infection group.[Conclusions] The UPEC rat orchitis model was successfully constructed;whether at the gene level or Protein level,UPEC Promoted the increase of Ca SR expression in TM.[Purpose]: Based on UPEC Promoting the increase of Ca SR expression in testicular macrophages,we further studied the role of Ca SR in the mechanism of orchitis and explored the natural immune mechanism of infective orchitis.[Methods]: Construct UPEC rat orchitis model and divide into normal saline Control group,UPEC infection group,Ca SR inhibitor NPS2143 negative Control group and UPEC +NPS2143 inhibitor group.On the first day,we constructed 10 normal saline Control rats and 20 UPEC infected groups according to the experimental method in the first Part.The next day,10 rats in the UPEC infection group were taken and the Ca SR inhibitor NPS-2143 was orthotopically injected into the testis to construct the UPEC + NPS2143 inhibitor group.Then 10 equal saline DMSO(NPS-2143 solvent)was injected in situ into the testes of 10 normal saline Control group and 10 UPEC infected group rats,and another 10 NPS-2143 negative Control groups were constructed.Observe the histological changes of the testis;take the epididymal sperm to analyze the total number of sperm and forward movement;use mass spectrometry to detect and analyze the level of testosterone in the serum of each group of rats;we use fluo-am fluorescent labeling flow method And the microplate reader method to detect the calcium ion in the macrophages of each group of rats;the ELISA method was used to detect the secretion of IL-1β in the testicular interstitial fluid and the primary TM supernatant;then,in vitro experiments,first normal rat testis macrophages were isolated,and then stimulated with bacterial UPEC in cell culture medium for 0min,30 min,60min,120 min in vitro to detect the gene expression levels of Ca SR and NLRP3 in TM after UPEC stimulation.At the same time,in vivo experiments,the primary TM was isolated and the expression levels of Ca SR and NLRP3 m RNA in TM were detected;Western Blot experimental method was used to detect the expression of NLRP3 and Caspase-1 Protein in the primary TM.[Results]: The testicular tissue sections of the UPEC infected group showed obvious inflammatory damage,including disordered spermatogenic epithelial arrangement,spermatogenic cells with vacuolar-like changes,and infiltration of inflammatory cells in the interstitial;Inflammation of the testes in the NPS-2143 inhibitor group was alleviated;there was no significant change in the NPS-2143 negative Control group compared with the saline Control group.Compared with the Control group,the total sperm count and sperm forward movement rate of rats after UPEC infection decreased significantly,while the total sperm count and sperm forward movement rate increased after using Ca SR inhibitor NPS-2143.Under normal circumstances,the level of testosterone in rat serum is about4.5ng / m L,and the level of testosterone in rats decreases about 3 times after UPEC infection.After intervention by Ca SR inhibitor NPS-2143,the level of testosterone compared with the UPEC infection group About doubled.In vivo experimental flow cytometry results showed that compared with the Control group,the UPEC group of Primary macrophages contained a large amount of calcium influx in the cytoplasm(P<0.01),and the calcium influx decreased after using the Ca SR inhibitor NPS-2143(P<0.05).The results of microplate reader detection of macrophages cytoplasmic calcium ion level showed that the level of calcium ion in UPEC + NPS-2143 group was lower than that in UPEC infection group(P <0.05).The results of in vitro experiments showed that the optimal time for bacterial UPEC stimulation in vitro was 60 min.The gene levels of Ca SR and NLRP3 in TM increased significantly after UPEC stimulation.Western Blot results showed that the Protein level of NLRP3 in primary testis macrophages of rats was significantly increased after UPEC infection,while Ca SR inhibitors can inhibit the expression of NLRP3 Protein.[Conclusion]: Abnormally increased Ca SR is one of the important factors that cause UPEC testicular inflammatory damage,which has a negative impact on sperm motility,total sperm count,testicular structure and serum testosterone levels.However,the Ca SR inhibitor NPS-2143 can be to a certain extent Relieve these inflammatory injuries;after UPEC infection,Ca SR in testicular macrophages may activate the NLRP3 inflammatory corpuscle Pathway by Promoting calcium influx.[Purpose]: In this part,we explore the role of Ca SR between PK2 and NLRP3 based on the activation of NLRP3 inflammatory corpuscle Pathway by PK2 in testicular macrophages,resulting in increased expression of IL-1β.We speculate that PK2 may activate the NLRP3 inflammatory corpuscle Pathway by regulating calcium ions.Further improve the mechanism of natural immunity of orchitis.[Methods]: Firstly,the UPEC rat orchitis model was constructed and divided into a saline Control group,a UPEC infection group,a PK2 inhibitor PKR-A negative Control group and a UPEC + PKR-A inhibitor group,10 Wistar rats in each group.Referring to the experimental method of constructing the model in Part II,the UPEC + NPS2143 inhibitor group refers to the in situ injection of the PK2 inhibitor PKR-A in the testes of UPEC-infected rats the next day,in which PKR-A is dissolved with 5% DMSO,The remaining groups of model mice were injected with equal volume of DMSO in situ.After the orchitis model was established,primary testicular macrophages were isolated.Fluorescence localization of PK2 and Ca SR in primary testis macrophages.Observe the changes in testis histology of each group;use mass spectrometry to detect and analyze the level of testosterone in the serum of rats in each group,and extract primary mesenchymal cells at the same time,by the following groups(Control,IL-1β,LPS + UPEC,LPS + UPEC+ PK2,PK2,anti-IL-1β)stimulate interstitial cells,and test the levels of testosterone in each group.The secretion of IL-1β in testicular interstitial fluid of each group was detected by ELISA.After treating primary testis macrophages with NLRP3,PK2,Caspase-1 and Ca SR inhibitors respectively,the supernatant of cells was collected to detect the expression of IL-1β,which included Ctrl,LPS,DMSO,LPS + UPEC + DMSO,LPS + UPEC +NPS2143,LPS + UPEC + MCC950,LPS + UPEC + VX-765 and LPS + UPEC + PK2 +DMSO,LPS + UPEC + PK2 + NPS2143,LPS + UPEC + PK2 + MCC950,LPS + UPEC +PK2 + VX-765 group.The level of PK2 of interstitial cell line(TM3)was overexpressed by Plasmid transfection,and the expression level of Ca SR Protein after PK2 overexpression was detected by Western Blot experiment method.[Results] PK2 is also expressed in the nucleus of primary testis macrophages,and UPEC stimulates the secretion of PK2 from the nucleus into the cytoplasm.The ELISA results of IL-1β showed that the content of IL-1β could not be detected in the supernatant of the Control group.The secretion level of IL-1β in the cell supernatant of the UPEC group was significantly increased.However,the IL-1β in the PKRA and NPS2143 groups Secretion levels show a decline.Under the action of three inhibitors,increasing the level of PK2,the inflammatory effect also increases with the level of PK2,and NPS2143 can inhibit the secretion of inflammatory cytokine IL-1β,but NPS2143 inhibits the efficiency of inflammatory cytokine IL-1β It did not increase with the increase of PK2.NPS2143 inhibited PK2 to Promote IL-1β secretion.The results of LC-MS in rat serum show that,consistent with the effect of Ca SR inhibitors,the use of PKR-A inhibitors can also Promote the Production of testosterone;the results of in vitro tests LC-MS show that testosterone can be detected in the Control group However,in some treatment groups(100 n M IL-1β,LPS + UPEC,LPS + UPEC + 10 n M PK2),the testosterone content in the supernatant was below the detection limit,and no corresponding results were shown.The total Protein WB extracted from PK2 transfected cells showed that the expression of Ca SR Protein in cells overexpressing PK2 was significantly increased.[Conclusion] PK2 can further Promote the expression of related factor Protein in NLRP3 inflammatory corpuscle Pathway in testicular macrophages,and induce testicular macrophages to secrete more IL-1β;PK2 induces NLRP3 inflammation after UPEC infection through Ca SR The activation of sex bodies,thereby Promoting the maturation and secretion of the Pro-inflammatory cytokine IL-1β. |
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