Studies On The Regulation Of Innate Immunity To Mitigate Cryptosporidium Parvum Infection With Curcumin And Chitosan

Cryptosporidium parvum is the second most common cause of diarrhea in children and animals,which can cause death,and is associated with long-term growth retardation and cognitive deficits in children.Nitazoxanide(NTZ)is currently the only FDA-approved drug for cryptosporidiosis,but this drug is not effective against immunocompromised and susceptible hosts such as infants and young children.Halofuginone is also approved in some countries for the control of infection in animals,especially calves and lambs,however,this drug is also ineffective for the treatment of Cryptosporidium infections in immunocompromised hosts.Therefore,there is an urgent need to find safe and effective drug for the treatment of cryptosporidiosis.In this study,the therapeutic effects of curcumin and chitosan on Cryptosporidium infection were observed,and their regulation of host innate immunity and influences on the structure and composition of gut microbiota were explored.In order to find effective drug against C.parvum infection,this study provides a basis for the treatment of cryptosporidiosis with curcumin and chitosan and its mechanism of action.One-week-old specific pathogen-free(SPF)neonatal immunodeficient C57BL/6 mice infected with1×10~6oocysts of C.parvum orally were examined for daily feces.Curcumin with a dosage of 4.33mg/kg/day was started after the onset of Cryptosporidium oocysts till days 15 post-infection(D15PI).We found that curcumin significantly(p<0.01)reduced the excretion of C.parvum oocysts in the feces of infected immunosuppressed neonatal mice by 74.70%,and prevented the reoccurrence of the second peak of oocysts excretion.Pathological section observation showed that curcumin supplementation also prevented epithelial damage,and villi degeneration,and no C.parvum oocysts were seen at the brush border compared to infected-untreated group of mice that reveals epithelial damage and the presence of oocysts at the brush border.16S r RNA sequencing analysis showed that curcumin significantly improved the gut microbial population structure,abundance and diversity in the infected group of mice.Curcumin supplementation increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Proteobacteria in mice infected with C.parvum.The relative abundance of Lactobacillus,Bacteroides,Akkermansia,Desulfovibrio,Prevotella,and Helicobacter was significantly associated with C.parvum infection inhibited by curcumin.Curcumin supplementation significantly(p<0.01)suppressed cytokines such as interferon-gamma(IFN-γ),interleukin-18(IL-18),and chemokines CCL2,CCL5,CXCL1,and CXCL10 gene expression levels in immunosuppressed neonatal C.parvum-infected mice.In vitro studies have shown that curcumin inhibited the invasion and intracellular growth of C.parvum on HCT-8 cells.Under the action of 200μM curcumin,the invasion rate of C.parvum sporozoites to HCT-8 cells decreased by 87.86%.Under the action of 200μM for 48 h,the growth inhibition rate of C.parvum in HCT-8 cells was as high as 93%.To revealed the mitigation effects of chitosan against C.parvum infection,one hundred and sixty SPF C57BL/6 juvenile mice(around 3 weeks old)were immunosuppressed and randomly divided into 5 groups(n=32 per group).The unchallenged mice received a basal diet(control)and three groups of mice challenged with 1×10~6C.parvum received a basal diet,a diet supplemented with 50 mg/kg/day paromomycin,1 mg/kg/day chitosan,and unchallenged mice treated with 1 mg/kg/day chitosan.Chitosan supplementation regulated serum biochemical indices and significantly(p<0.01)75.54%reduced C.parvum oocyst excretion in infected mice treated with chitosan compared with the infected mice that received no treatment.Microbial diversity indices were decreased but not significantly affected by C.parvum or supplemental chitosan.However,microbial community richness in the C.parvum-infected group that received no drug treatment decreased significantly compared to the infected group supplemented with chitosan(p<0.05).Moreover,chitosan supplementation also increased the relative abundance of Bacteroidetes/Bacteroides,while that of Proteobacteria,Tenericutes,Defferribacteres,and Firmicutes decreased(p<0.05).Chitosan-fed infected mice showed significantly(p<0.01)decreased m RNA expression of IFN-γand TNF-αcompared to infected mice that received no treatment.Furthermore,chitosan inhibited induction of the TLR4 signaling pathway.Oral supplementation of chitosan significantly(p<0.01)inhibits TLR4 expression in intestinal tissue of C.parvum infected mice when compared with infected mice that received no drug treatment.Moreover,chitosan also significantly upregulated STAT1protein expression(p<0.01)in C.parvum-infected mice compared to infected mice group without chitosan supplementation.In summary,this study found that curcumin and chitosan can effectively reduce the excretion of oocysts in the feces of C.parvum infected mice.The research results help to further understand the anti-Cryptosporidium mechanism of curcumin and chitosan,and provide a basis for finding effective anti-Cryptosporidium drug.