| Objective To observe the impacts of free rhubarb anthraquinones on specific and non-specific immune function in intestinal lymphatic system of SAP rats and to investigate the variations of intestinal flora and relations with immune function in the intestinal lymphatic system.Methods(1)A total of 18 healthy male SD rats were randomly divided into control group,SAP model group and free rhubarb anthraquinones intervention group.MLN was collected 24 hours after molding and the intestinal tissue was given the paraffin embedding.Besides,partial MLN was collected to prepare cell suspension.The histopathological changes of MLN were observed under light microscope after HE staining.Expressions of NLRP3 and ASC were tested in the MLN and intestine respectively by immunoflurescence method.The expressions of cysteine,Caspase-1 and SIgA were tested in the MLN and intestine by immunohistochemistry.Th1/Th2 ratio and Treg percentage of Foxp3 marker of MLN cell suspension were tested by flow cytometry.(2)A SAP rat model was made by slowly injecting 3.5% sterile injection of sodium taurocholate using retrograde cholangiopancreatography.A total of 18 healthy male SD rats were randomly divided into control group(C),SAP model group(M)and free total rhubarb anthraquinones intervention group(F).The anus was disinfected by iodophor 24 hours after molding.Rat’s faeces was collected by EP tube,cryopreserved at-80 ℃,and delivered to TinyGeneBio-Tech(Shanghai)Co.,Ltd using an ultralow temperature ice bag for microflora analysis.Results(1)All rats’ s predominant bacteria are the same,including bacteroidetes,proteobacteria and firmicutes.The relative abundance of bacteroidetes and firmicutes is higher while the relative abundance of proteobacteria is lower in group C.Compared to group C,the relative abundance of firmicutes and bacteroidetes is significantly decreased while the relative abundance of proteobacteria is significantly increased in group M.The relative abundance of firmicutes and bacteroidetes is improved compared to group M while the percentage of relative abundance of proteobacteria is reduced to some extent.The diversity in group M is significantly decreased compared to group C while the bacterial diversity in group F is increased to some extent.(2)Small intestine and MLN histopathological changes have been investigated under light microscope.Rat’s small intestinal mucosa epithelium has appeared the degeneration and necrosis in group M,as well as severe villus falling off,angiectasis in the mucosa and under the mucosa,bleeding a lot and inflammatory cell infiltration.Small intestinal lesions are eased apparently in group F,with partial villus fusion and defects,hyperemia and edema in the lamina propria,and a little inflammatory cell infiltration.Lymphoid nodules appear to be enlarged,cortex is thickened,the quantity of lymphoid nodules is increased in the cortex area in group M compared to group C.The quantity and size of lymphoid nodules in the cortex area are smaller in group F than group M.IFtest shows that the expressions of and NLRP3 and ASC in rat’s small intestine and MLN are up-regulated in group M.Expressions of NLRP3 and ASC are down-regulated in group F.IHC test shows that the expression of Caspase-1in rat’s small intestine and MLN is up-regulated in group M.The expression of Caspase-1 is down-regulated in group F.Moreover,the expression of SIg A is significantly down-regulated in group M while the expression of SIgA is up-regulated to some extent in group F.FCM test shows that Th1 of MLN in group M is significantly up-regulated compared to group C(P<0.05)while it is down-regulated in group F compared to group M(P<0.05).Th2 in group M is significantly down-regulated compared to group C(P<0.05)while it is significantly up-regulated in group F compared to group M(P<0.05).In comparison of Th1/Th2,it is significantly up-regulated in group M compared to group C(P<0.05)while it is down-regulated in group F compared to group M(P<0.05).CD4~+CD25~+Foxp3~+ in group M is obviously higher than that of group C(P<0.05)while it is obviously lower in group F than group M(P<0.05).The percentage of Treg in group M is apparently higher than that of group C(P <0.05)while it is apparently lower in group F than group M(P<0.05).(3)In non-specific immunity,bacterial diversity is significantly negatively correlated(P <0.01)to expressions of NLRP3,ASC,caspase-1,Th1 and Treg in the small intestine and MLN while it is significantly positively correlated(P <0.01)to expressions of SIgA and Th2.The quantity of proteobacteria is significantly positively correlated(P <0.01)to expressionsof NLRP3,ASC,caspase-1,Th1 and Treg in the intestine and MLN while it is significantly negatively correlated(P <0.01)to expressions of SIgA and Th2.The correlation of bacteroidetes and firmicutes with immune response in the intestinal lymphatic system is not significant.Conclusion The intestinal flora disorders such as intestinal bacterial diversity changes and the surge in quantity of proteobacteria have happened in the early stage of SAP.Massive bacterial endotoxin is produced and will induce extreme immune response in the intestinal lymphatic system.Excessive,inherent and specific immune response generates a large number of inflammatory damage factors which will cause multiple organ injuries via lymph vessels and distant shifting.Free total rhubarb anthraquinones may play a protective role in multiple organs by improving intestinal flora imbalance and then inhibiting extreme immune response in the intestinal lymphatic system. |
PDF Full Text Request