| Toxoplasma gondii(T.gondii)is a specific intracellular protozoan,which infects most warm-blooded animals including humans,and is one of the most prevalent zoonoses.T.gondii can cause abortion of pregnant animals,stillbirths or vulnerable weak fetuses,and reproductive dysfunction of breeding animals,affecting the development of animal husbandry and causing huge economic losses.At the same time,eating pigs,cattle,sheep and other meat is an important way for human infection with T.gondii.T.gondii in pregnant women often causes abortion,congenital infection of neonates with intracranial calcification,chorioretinitis,hydrocephalus and mental retardation.Therefore,T.gondii has become one of the main factors which endanger the normal reproduction of livestock and eugenic breeding of human beings.T.gondii can infect almost any nucleated cell in the host and replicate,leading to toxoplasmosis.T.gondii can cause placenta damage in pregnant animals.T.gondii can cause pathological damage of testis,epididymis,vas deferens,prostate and other reproductive organs and influence sperm formation and function in male animals.T.gondii can cause hepatomegaly,hepatitis,hepatic granuloma,hepatic necrosis,cholestasis jaundice and cirrhosis liver and other clinical symptoms.Drugs such as sulfadiazine and pyrimidine have been used to treat toxoplasmosis effectively,but these compounds can cause serious side effects.Therefore,the development of safe and effective anti-T.gondii drugs is a hot research topic at home and abroad.T.gondiiinfected pregnant mice model,male mice model and hepatic injury mice model were established to study the anti-T.gondii effect and mechanism of polysaccharide from Inonotus obliquus.Methods:(1)In the model of pregnant mice infected with T.gondii,72 pregnant mice(pregnancy for 10 days)were randomly divided into six groups(n=12):normal group,T.gondii group,T.gondii+IOP 100 mg/kg group,T.gondii+IOP 200 mg/kg group,T.gondii+IOP 400 mg/kg group and T.gondii+sulfadiazine 200 mg/kg group.T.gondii(RH strain,103 strains/mice)was injected into the abdominal cavity of mice except the normal group.Mice in IOP and sulfadiazine group were given IOP or sulfadiazine by gavage.On the 17th day of gestation,the pregnant mice were executed,blood was collected,and the placenta and uterus were separated and stored in-80℃ refrigerator.In this study,the pregnancy outcome of pregnant mice was evaluated.The contents of malondialdehyde(MDA),superoxide dismutase(SOD)and glutathione(GSH)in placenta of pregnant mice were determined.The expression of progesterone(P),estriol(E3),tumor necrosis factor-alpha(TNF-α),interleukin-6(IL-6),interleukin-1β(IL-1β),interferon-gamma(IFN-γ),interleukin-17A(IL-17A)and interleukin-10(IL-10)in serum and placenta of pregnant mice were measured by ELISA.The expression of IL17A and transforming growth factor-beta(TGF-β)in placenta was determined by ELISA.The expression of IL-6,IL-17A,transforming growth factor-β(TGF-β),orphan receptor gamma t(RORyt)and fork head protein P3(Foxp3)RNA in placenta tissue were detected by RT-PCR.The expression of Foxp3,signal transduction and transcription activator 3(STAT3),RORyt,Toll-like receptor 4(TLR4),nuclear factorkappa B(NF-κB)p65 and phosphorylation inhibitor kappaB alpha(IκBα)proteins in placenta tissue was detected by immunoblotting.(2)72 male BALB/C mice were randomly divided into six groups:normal group,T.gondii group,T.gondii+IOP 100 mg/kg group,T.gondii+IOP 200 mg/kg group,T.gondii+IOP 400 mg/kg group and T.gondii+Sulfadiazine 200 mg/kg group.T.gondii(RH strain,103 strains/mice)was injected into the abdominal cavity of mice except the normal group.Mice in IOP and sulfadiazine group were given IOP or sulfadiazine by gavage for 7 consecutive days.After the last administration,the mice were anesthetized with ether.Blood was collected and testicular tissue was fixed in formalin or stored in-80℃ refrigerator.In this study,sperm parameters were evaluated.Serum testosterone(T),luteinizing hormone(LH)and follicle stimulating hormone(FSH)levels were measured by ELISA.The expression of steroid-producing acute regulatory protein(StAR),cytochrome P450 cholesterol side chain lyase(P450scc)and 17βhydroxysteroid dehydrogenase(17β-HSD)which were key genes of testosterone synthesis in testicular tissues were determined by RT-PCR.Apoptosis of spermatogenic cells was detected by TUNEL.Immunoblotting were used for decteding the expression of Bax,Bcl-2 and Cleaved-caspase 3 in testis.(3)72 male BALB/C mice were randomly divided into six groups including normal group,T.gondii group,T.gondii+IOP 100 mg/kg group,T.gondii+IOP 200 mg/kg group,T.gondii+IOP 400 mg/kg group and T.gondii+Sulfadiazine 200 mg/kg group.T.gondii(RH strain,103 strains/mice)was injected into the abdominal cavity of mice except the normal group.Mice in IOP and sulfadiazine groups were given IOP or sulfadiazine by gavage for 7 consecutive days.After the last administration,the mice were anesthetized with ether and blood was collected.The liver tissue was fixed in formalin or stored in-80℃ refrigerator.The liver coefficients,alanine aminotransferase(ALT),aspartate aminotransferase(AST),MDA,nitric oxide(NO),SODand GSH were measured.Serum TNF-α,IL-6,IL-1β,IFN-γ and interleukin-4(IL4)were detected by ELISA.Toll-like receptor 2(TLR2),Toll-like receptor 4(TLR4),p-NF-κB p65,p-IκBα,nuclear factor E2-related factor 2(Nrf2)and hemoglobin-1(HO1)protein expression were detected by immunoblotting.Results:(1)IOP can significantly reduce the abortion rate of pregnant rats caused by T.gondii infection.IOP can inhibit the decrease of serum P and E3 of pregnant mice infected with T.gondii.IOP can inhibit the expression of MDA and increase the contents of SOD and GSH in placenta in pregnant mice ELISA results showed that IOP can reduce the levels of pro-inflammatory cytokines TNF-α,IL-6,IL-1β,IFN-γ and IL-17A in serum in pregnant mice infected with T.gondii,and increase the levels of antiinflammatory factor IL-10.ELISA results showed that IOP could inhibit the secretion of IL-17A and increase the content of TGF-β in placenta in pregnant mice infected with T.gondii.RT-PCR results showed that IOP inhibit the decrease of Foxp3,TGF-β and the increase of IL-6,IL-17A and RORyt in placenta tissue in pregnant mice caused by T.gondii infection.Immunoblotting results showed that IOP significantly increased the expression of Foxp3 and decreased the expression of STAT3,TLR4,p-NF-κBp65,pIκBα in pregnant mice infected with T.gondii.(2)IOP could inhibit the decrease of sperm number and increase of sperm heteromorphism rate caused by T.gondii infection.IOP could decrease the serum T,LH and FSH contents in mice infected with T.gondii.RT-PCR results showed that IOP promote the expression of StaR,P450scc and 17 β-HSD in male mice infected with T.gondii.TUNEL results showed that IOP inhibit testicular germination in mice infected with T.gondii.Apoptosis of spermatogenic cells and the expression of apoptosisrelated proteins Bax,Bcl-2 and Cleaved-caspase 3 were also regulated.(3)IOP can inhibit the elevation of ALT and AST in mice induced by T.gondii.IOP can improve the liver injury caused by T.gondii infection.IOP can inhibit the expression of inflammatory cytokines TNF-α,IL-6,IL-1β,IFN-γ and IL-4 in mice serum.IOP can promote the expression of antioxidant enzymes SOD and GSH,and reduce the content of MDA.IOP can inhibit the activation of TLR2,TLR4,the phosphorylation of NF-κBp65,Nrf2 nuclear transfer and enhanced HO-1 activity in mice liver tissue.Conclusion:(1)IOP can resist the placental damage caused by T.gondii infection and maintain the normal pregnancy of pregnant mice.IOP can inhibit the Th17/Treg imbalance induced by T.gondii by reducing Th17 cell-related cytokines and increasing Treg-related cytokines expression.IOP may change the balance of Th17/Treg cells through TLR4/NF-κB pathway to protect the placenta of pregnant mice from T.gondi-induced damage.(2)IOP can protect male mice infected with T.gondii from reproductive damage.IOP can inhibit the down-regulation of StAR,P450scc and 17β-HSD expression in testicular tissue caused by T.gondii infection,thus antagonizing the decrease of testosterone caused by T.gondii infection.IOP can resist the apoptosis of testicular cells caused by T.gondii infection by regulating the expression of apoptosis-related proteins Bax,Bcl2 and Cleaved-caspase3.(3)IOP has a good regulatory effect on liver injury caused by T.gondii infection.IOP regulated the production of serum inflammatory cytokines by regulating TLRs/NF-κB signaling pathway to resist liver inflammation caused by T.gondii infection,and regulated lipid peroxides and antioxidant enzymes by activating Nrf2/HO-1 signaling pathway to resist liver oxidative stress caused by T.gondii infection. |
