| Bovine mastitis is one of the most frequently-occurring diseases causing great economic losses to worldwide dairy cow breeding,which also severely threatens human food safety.The present study aimed at exploring the mechanism of bacteriostasis of curcumin against mouse mastitis model induced by Staphylococcus aureus(S.aureus)and its anti-inflammatory effects through systemic in vitro and in vivo experiments in the level of cell and cellular,and providing sufficient experimental and clinical data and lay essential and theoretical foundation for the application of Mongolian veterinary drug of Curcuma Pulvis that majorly consists of curcumin.In Chapter one,in vitro bacteriostasis of curcumin,Curcuma Pulvis and antibiotics against mastitis-induced S.aureus was verified with Disc diffusion test and broth dilution method.Enzyme-labeled Turbidimetry,microbial adhesion method and detection kits were applied to measure the influences of curcumin and Curcuma Pulvis on the growth curve,cell surface hydrophobicity and permeability of cell wall and membrane of S.aureus.In Chapter two,the mechanism of curcumin and Curcuma Pulvis in mouse mastitis model induced by S.aureus isolated from bovine milk.Two hundred and fifty mice were equally divided into five groups and separately reckoned as blank control group,model group,antibiotic-treated group(positive control group),Curcuma Pulvis-treated group and curcumin-treated group.The latter four groups were all mastitis-modeled mouse,and the latter three groups were separately treated with 0.13 g/kg of Ciprofloxacin or 1.0 g/kg of Curcuma Pulvis via drenching or 0.125g/kg of curcumin via intraperitoneal injection.Every kind of treatment was conducted two times each day for five continuous days.Histopathological sections and hematoxylin-eosin staining were applied to observe histopathological changes of structure of MMECs and mammary tissues.Flow cytometry,Enzyme-Linked Immunosorbent Assay and MTT method were used to detect the content of lymphocyte subtypes and cytokines in serum of mouse.In Chapter three,enzyme digestion method and differential pasting method were combined together to purify MMECs,and the influences on TLR2-mediated NF-?B signal pathway in MMECs induced by curcumin were verified.The results were as followed:In Chapter one,curcumin and Curcuma Pulvis showed satisfactory bacteriostasis especially with No.1 minimum inhibitory concentration,which brought about the best bacteriostasis against 1×107 cfu/mL of S.aureus(P<0.05).Both curcumin and Curcuma Pulvis promoted the advance of the decline phase of S.aureus and obviously magnified the permeability of cell wall and membrane,namely,showing bacteriostasis.In Chapter two,the influence on the morphological structure of mammary gland in mastitis-modeled mouse by S.aureus isolated from bovine milk could be observed as followed:normal acinar structure reduced;desmoplasia appeared;mild infiltrations of polymorphonuclear neutrophils(PMNs)into interlobular duct.However,the morphological structure of mammary gland in mastitis-modeled mouse treated with curcumin or Curcuma Pulvis showed increased amount of lymphocytes and decreased infiltrations of PMNs in the alveoli.Furthermore,the amounts of desmocyte in the alveoli gradually increased and connective tissue shaped by degrees,which demonstrated that the structure of alveoli was partly repaired.The organ index of groups treated with curcumin,Curcuma Pulvis or Ciprofloxacin was significantly higher than that of negative group(P<0.05).Compared with immune responses in mice of negative control group,the serous content of CD8+,TNF-?,IL-6,IL-1 and IL-8 were obviously depressed(P<0.05),and the serous content of IFN-y,IL-2 and IL-10 were obviously increased,the content of CD4+T cells,CD3+T cells and CD19+B cells were increased,and the cytoactive of Natural killer cell(NK cell)were strengthened,which stands for the improvement of anti-inflammatory immunity by curcumin,Curcuma Pulvis or Ciprofloxacin.In Chapter three,cytokeratin 8/18 of purified epithelial cells were positive.1.0×106 cfu/mL of S.aureus resulted in the best inflammatory reactions with a working time of 24 hoxurs.And 20?g/mL of curcumin showed the best inhibitory effects with a working time of 12 hours.In addition,curcumin significantly decreased the expression of inflammatory factors including IL-1?,IL-6 and TNF-a via regulating signal pathway(P<0.05).Conclusion was drawn as followed:curcumin and Curcuma Pulvis showed pretty satisfactory functions including significantl inhibition on in vitro growth of S.aureus,obvious improvement on organ index of mastitis modeled mouse,reinforcement on viability of lymphocytes and its subgroups and cytoactivity of NK cells,regulation on NF-?B signal pathway and enhancement on non-specific immune responses and anti-inflammatory effects.The present research provides important basement for the application of Curcuma Pulvis as Mongolian drugs for the prevention and therapy of S.aureus-induced mastitis. |
PDF Full Text Request