Effect Of Polysaccharide Nucleic Acid Fraction Bacillus Guerin On Endotoxin Induced Experimental Animal Mastitis

In this study,effects of BCG-PSN on mammary defense and protective effects of BCG-PSN on mammary gland were first evaluated by experimental mastitis induced by LPS in goats.Then,the mechanism by which BCG-PSN activated natural defense system was discussed in rat model.On the base of these,changes of PMN function were observed after co-incubation with BCG-PSN.The results as follows:1 Effect of LPS on Inflammation Factors in Mammary Tissue of Goats Relating with MastitisEight healthy Suining white goats at early stages of lactation were infused with endotoxin or lipopolysaccharide(LPS)(50μg/bwt) in the left half of the mammary gland and physiological saline was infused in the control contralateral half.At intervals of 0,1,2, 3,4,5,6,7,8,9 and 24 h after infusion,mediators of inflammation were measured and the degree of neutrophil(PMN) infiltration and clinical variables were recorded.After 24 h,the animals were euthanized and mammary tissue was collected.A section was fixed for histopathologic evaluation and the remaining tissue was stored at -70℃.Clinical and histopathologic examination indicated that infusion of LPS induced acute mastitis in left half of the mammary gland.Infusion of LPS significantly increased the activity of N-Acetyl-β-D-glucosaminidase(NAGase),myeloperoxidase(MPO) and inducible nitric oxide synthase(iNOs) and the concentration of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β) and interleukin-6(IL-6)(P＜0.01).There was a general increase in the levels of mRNA cytokine expression,too.The result showed that over release of inflammatory factors may cause damage to mammary gland.2 Protection of BCG-PSN to Mammary Tissue of Goat Experimental MastitisSix healthy Suining white goats at early stages of lactation were intramammary infused with BCG-PSN(5mL) in the left quarters and physiological saline(5mL) in the control contralateral quarters.The administration of polysaccharide nucleic acid of Bacillus Guerin (BCG-PSN) was carried out for six consecutive days.On the seventh day,the mammary quarters(both right and left) of the six(6) goats were infused with LPS(50μg/bwt).At varying intervals of 0,1,2,3,4,5,6,7,8,9 and 24 h before and after infusion inflammation was recorded.After 24 h,the animals were euthanized and mammary tissue was collected from the same position.A section was fixed for histopathological evaluations and the remaining tissue was stored at -70℃.Clinical and histopathological examinations indicated that infusion of LPS induced acute mastitis in both quarters.The results showed that infusion of BCG-PSN significantly reduced activity of NAGase,MPO,iNOS and the concentration of TNF-α,IL-1β,and IL-6(P＜0.05).There was a general reduction in the levels of mRNA cytokine expression(P＜0.05).These findings suggest that BCG-PSN inhibits release of inflammatory factors that cause damage to mammary epithelial cells during mastitis infections in goats.3 Protection of BCG-PSN to Mammary Tissue of Rat Experimental MastitisThe aim of this study was to evaluate in rats,protection of BCG-PSN to mammary tissue of rat experimental mastitis.Seventy-two lactating SD rats were randomly divided into control and treatment group(n=36).BCG-PSN(treatment group,TG) or sterile pyrogen-free physiological saline(control group,CG) 0.33ml were injected into the left tibialis anterior muscle of rats 14 days after conception.The administration was repeated for five consecutive times every other day.Seventy-two hours after parturition,30 rats of each group were inoculated with 10μg Escherichia coli LPS dissolved in 100μL physiological saline into the left 4(L4) and right 4(R4) mammary glands and six as control. Just before the inoculation(control group defined as 0 h) and at 3,6,9,12 and 24 h after inoculation six rats at every time point were euthanatized and samples were collected.The histopathological observation indicated that treatment with BCG-PSN made a rapid renovation of mammary tissue compared with CG.Milk secretion elevated with an increasing of the luminal spaces with milk at the later stage of inflammation in TG.In mammary tissue,both NAGase and iNOs peaked at 6 h in TG and 9 h in CG,whereas serum NAGase reached nadir level at 12 h PI in TG and 9 h PI in CG,and serum iNOs reached nadir level at 6 h and 3 h PI respectively.Administration BCG-PSN reduced the peak level of TNF-α,IL-1βand IL-6 in mammary tissue compared with CG,whereas no significant changes were observed in serum after LPS challenge.RT-PCR analysis showed that BCG-PSN decreased the toll-like receptor-4(TLR-4) mRNA expression level from 3 h PI and significant reduction was observed at 6h,9 h and 24 h PI.These findings suggested that BCG-PSN has a general protective effect against LPS-induced local mammary injury by reducing TLR-4 mRNA expression level and further inhibiting the release of inflammatory factors that cause damage to mammary gland.4 Immunoregulation of BCG-PSN to Rats of Experimental MastiffsThe aim of this study was to evaluate in rats,immunoregulation of BCG-PSN to rats of experimental mastitis.Seventy-two lactating SD rats were randomly divided into control and treatment group(n=36).BCG-PSN(treatment group,TG) or sterile pyrogen-free physiological saline(control group,CG) 0.33ml were injected into the left tibialis anterior muscle of rats 14 days after conception.The administration was repeated for five consecutive times every other day.Seventy-two hours after parturition,30 rats of each group were inoculated with 10μg LPS dissolved in 100μL physiological saline into the left 4(L4) and right 4(R4) mammary glands and six as control.Just before the inoculation (control group defined as 0 h) and at 3,6,9,12 and 24 h after inoculation six rats at every time point were euthanatized and samples were collected.The results indicated that the concentrations of MPO in mammary tissue peaked at 6 h in TG,and in CG kept a high level until 24 h PI.Significant reduce of MPO was observed at 12 h and 24 h PI compared TG with CG.Serum MPO reached nadir at the same time(12 h after LPS challenge) both CG and TG.After LPS infusion,the concentrations of IL-2 increased dramatically and peaked at 24 h both CG and TG.BCG-PSN induced significant elevated of IL-2 at 0 h and 24 compared with control.Flow cytometfic analysis showed that,compared with control(0 h),the 24 h group exhibited a CD4+(T-helper) T cell decline,a CDS+(T-cytotoxic or T-suppressor) T cell increase,and decreased CD4/CD8 ratio in CG.No significant changes were observed in TG.The results of RT-PCR showed that in CG,the levels of IL-2,and INF-γmRNA expression increased,whereas IL-4 mRNA expression decreased after LPS challenged.As a consequence,the INF-γ/IL-4 mRNA radio was significant higher 3 h,6 h, and 9 h PI compared with normal value(0 h).BCG-PSN increased the mRNA expression both INF-γand IL-4 before infusion of LPS.After LPS challenged,significantly reduced Th1/Th2 cytokine radio was observed,because Th1 cytokine IFN-γwas suppressed and Th2 cytokine IL-4 was enhanced compared with CG.The study demonstrated that BCG-PSN may protect the mammary gland by improving the non-special and special immune factors of rats.5 Effect of BCG-PSN on Phagocytosis and Generation of Reactive Oxygen Species of Lactating Cow Peripheral Blood PMNThe aim of this study was to evaluate in vitro,effect of BCG-PSN on phagocytosis and generation of reactive oxygen species(ROS) of lactating cow peripheral blood PMN. Peripheral blood of healthy lactation cow at early stages of lactation was collected from the jugular vein by venipuncture in evacuated tubes containing heparin as anticoagulant.(1) FITC-labeled E.coli 10μL was added to each sample after a 2 h co-incubation with different concentrations of BCG-PSN(final concentration 0,2,5,10,40,80μg/mL).After further 20 min incubation at 37℃,phagocytosis was stopped by dipping into ice water. Then erythrocytes were lysed with lysing solution and the leucocytes were washed two times with PBS(0.01mol/L,pH7.2).The cells were then resuspended in PBS and analyzed on a FACScan flow cytometer.FL1 green channel captured FITC fluorescence.(2) Phorbol myrisate acetate(PMA,final concentrationl00ng/mL) was used to stimulate PMN after a 2 h co-incubation with different concentrations of BCG-PSN(final concentration 0,2,5,10,40,80μg/mL).Control tests of every group were set up using PBS instead of PMA.All tubes were incubated at 37℃for 15 min and then working dihydrorhodamine 123(DHR 123,final concentration 5μM) solution was added.After further 10 min incubation at 37℃, erythrocytes were lysed with lysing solution.Then the leucocytes were washed two times with PBS(0.01mol/L,pH7.2) and stabilized by paraformaldehyde.The the cells were then resuspended in PBS and analyzed on a FACScan flow cytometer.The results showed that BCG-PSN could significant reduced the generation of reactive oxygen species of PMN and had no effect on the phagocytosis of PMN.This suggests that BCG-PSN protects the mammary gland from inflammatory injury by attenuating the release of ROS but not by directly phagocytosis.BCG-PSN may be a potential candidate to prevent bovine mastitis.In conclusion,BCG-PSN may protect the mammary gland by modulating the secretion of non-special and special immune factors,down-regulating the expression of TLR-4 and inflammation mediator.The results suggest BCG-PSN can be used as a potential candidate to prevent mastitis.