| Objective:Immune disorder is an important reason for patients with pancreatitis to turn to severe or even death.Therefore,the timing of immune regulation has become a major issue for SAP treatment.Can rhubarb,the most commonly used Chinese medicine for the treatment of SAP,be used for SAP under different immune states?Previous studies have found that the intestinal mucosal immune system plays an important role in the immune disorder of SAP.This study intends to dynamically observe the changes in the intestinal mucosal immune system’s inflammatory/anti-inflammatory factors,the number of immune cells,and humoral immune factors,and observe the immunity of SAP rats Dynamic changes.On this basis,the difference in efficacy between Rhubarb and Rhubarb free anthraquinones(FTRAs)administered under different immune states was evaluated to determine the best timing of medication.Next,a sublethal dose of Pseudomonas aeruginosa was injected to simulate secondary infection to verify the protective effect of the best timing of medication.Methods:(1)SAP immunokinetic changes:(1)3.5%sodium taurocholate(Na Tc)prepared SAP rat model,observed the 14-day survival rate of the rat and the general condition of the pancreas at 1h,3h,6h,12h,24h,36h,48h,72h,120h,168h and 336h.HE staining observed the pathological changes of the pancreas,serum amylase and The pancreatic lipase activity level evaluates the severity of SAP.(2)Observe the pathological changes of the small intestine by HE staining,and detect the serum D-lactic acid level by ELISA to reflect the intestinal mucosal barrier function.(3)Detection of small intestinal homogenate IL-1β,TNF-α,IL-6,IL-8,IL-18(inflammatory factor),TGF-β,IL-10,IL-4,s TNF-αR(anti-inflammatory factor)by ELISA)Level,the limulus reagent quantitative method to detect plasma endotoxin level reflects the change rule of SAP intestinal inflammation.(4)The pathological changes of mesenteric lymph nodes(MLN)were observed by HE staining.The expression of CD68 and C103 in the small intestine was detected by immunohistochemistry to reflect the number of intestinal macrophages and dendritic cells.Flow cytometry was used to detect Th1,Th2,and Treg cells in the MLN.The ELISA to detect the content of SIg A in intestinal homogenate reflects the changes of humoral immunity in SAP rats.(5)Determination of SAP immune turning point:Determine the growth curve of Pseudomonas aeruginosa(PA)and determine the logarithmic growth period.Normal rats were injected with different doses of PA,the 7-day mortality was observed,and the sublethal dose of PA was screened.ELISA was used to detect the changes of IL-1βand TNF-αat 0h,2h,4h,6h and 12h after the sublethal dose of PA challenged by normal rats,and screen the best time point for sampling.ELISA was used to detect the changes in the levels of TNF-αand IL-1βin the intestines of rats given sublethal doses of PA at 0h,12h,24h,36h,48h after the establishment of SAP to determine the immune turning point.(2)To study the therapeutic effect of FTRAs on SAP in different immune states.Set up Sham group,SAP group,SAP+Rhubarb group,SAP+FTRAs(22.5mg/kg,45mg/kg,90mg/kg treatment group)before the SAP immune transition(0h),middle(48h),and later(72h)administration,12h/time,3 times in total,2h after the last administration,that is,24h,72h,96h after the model was put to death,the samples were taken.The detection indicators are the same as the first part.(3)To study the protective effect of FTRAs on the secondary PA infection in SAP rats,set up Sham group,SAP group,Sham+PA group,SAP+PA group,SAP+FTRAs+PA,SAP+FTRAs(22.5mg/kg,45mg/kg,90mg/kg)+PA,observe the 7-day mortality of rats,and detect intestinal IL-1β,TNF-α,IL-6,IL-8,IL-18(inflammatory factors)and TGF-β,IL-10,IL-4,s TNF-αR(anti-inflammatory factor)levels by ELISA,limulus reagent quantitatively detects plasma endotoxin levels.Results:(1)SAP immunokinetic changes:(1)The 14-day cumulative survival rate of SAP rats is 61.08%.Before 48h and after 48h,there are two death peaks of SAP;the pancreas is generally congested and necrotic after 1-6h after surgery,and the pancreas begins to sclerosis after 12h Yellow,adhesion to liver,intestinal tract and other organs.Pancreatic tissue dissolves necrosis in6-48h,and forms fibrosis and granulation tissue at 72-336h.Compared with the Sham group,the serum amylase activity gradually increased from 1-6h,reached the peak at 6h,remained basically unchanged at 6-36h,and gradually decreased after 36h.Pancreatic lipase activity increases and reaches its peak at 3-12h,and then gradually decreases.(2)In the SAP group,the intestinal mucosa shrank from 12h,the lamina propria inflammatory cells infiltrated,and the intestinal permeability increased.Compared with the sham operation group,serum D-lactate gradually increased from 12h,reached a peak at 48h,remained basically unchanged at 48-120h,and decreased slightly from 120-336h.(3)Compared with the Sham group,the intestinal IL-1β,TNF-α,IL-6,IL-8,IL-18,TGF-β,IL-10 and plasma endotoxin of rats in the SAP group were mainly obvious in 12-48h Increased and gradually decreased after 48h;IL-4 and s TNF-αR decreased significantly after 6-36h,and gradually increased after 36h.(4)The pathological changes of MLN were obvious at 3-336h,and the lymphatic follicles in the lymph nodes increased significantly.The pathological scores gradually increased at 3-12h,at a plateau at 12-36h,and the pathological scores decreased slightly at 36-336h.The expression of CD68 in the intestinal tract of rats in the SAP group increased slightly at 1-6h,significantly decreased at 12-72h,and gradually increased at 120-336h.The expression of CD103 in the intestinal tract gradually increased at 1-3h and gathered at the upper end of the villi.The expression of CD103 in the intestinal tract at 6-72h decreased significantly,and it gradually increased at 120-336h.The Th1 ratio of MLN in the SAP group gradually increased from 1 to 24h,at a plateau from 24 to 36h,began to gradually decline at 36h,and fell to a normal level at 72h.The Th2ratio did not change significantly from 1-36h,began to decline at 36h,and was at a plateau at 36-336h.The Th1/Th2 ratio did not change significantly in 1-3h,the ratio gradually increased at 6-24h,reached the peak at 24h,and then the ratio decreased gradually at 24-336h.The proportion of Treg cells in the MLN of the SAP group gradually increased at 12-24h,reached a peak at 24h,reached a plateau at 24-48h,decreased gradually at 48-168h,and returned to normal at168h.The intestinal humoral immune factor SIg A content of SAP rats gradually decreased from 1h,reached a trough at 3h,reached a low level plateau at 3-36h,gradually increased at 36-48h,and returned to normal level at 48-336h.The intestinal SIg A content remained basically unchanged.(5)Observing the growth curve of Pseudomonas aeruginosa(PA),it was found that the PA was in the logarithmic growth phase after 16 hours of culture;2.0×10~8CFU/kg PA was the sublethal dose;compared with the control group,after PA challenge,the intestinal IL-1βThe levels of TNF-αand TNF-αreached a peak at 2h,and then decreased,and 2h was the best time to collect materials after PA challenge.After the SAP model was established,sublethal PA was given.Compared with the SAP group,the intestinal IL-1βand TNF-αof rats in the SAP+PA group increased slightly at 0h,12h,24h,and 36h,but at 48h,compared with the SAP group In contrast,IL-1βand TNF-αwere slightly lower in the SAP+PA group.Therefore,it was determined that 48h was the turning point for SAP rats from the pre-excessive inflammatory response and mixed immune period to the compensatory immunosuppressive period.(2)Therapeutic effects of FTRAs on SAP in different immune states:(1)Administered before the immune transition,compared with the SAP-24h group,the pancreatic necrosis area of the SAP+FTRAs(45mg/kg,90mg/kg)-24h group was reduced,and the pancreatic damage was alleviated.FTRAs had no obvious effect when administered during and after the immune transition.Before the immune transition,compared with the SAP-24h group,the SAP+FTRAs(45mg/kg,90mg/kg)-24h group can inhibit the activity of SAP serum amylase and pancreatic lipase.There is no significant effect on the administration during and after the immune transition.(2)Through intestinal pathological observation,compared with SAP-24h group,SAP+FTRAs(45mg/kg,90mg/kg)-24h group can reduce the shedding of intestinal epithelial cells,reduce intestinal injury,and neutralize the immune transition.After the immune transition,FTRAs has no obvious effect.Compared with the SAP-24h group,the SAP+FTRAs(45mg/kg,90mg/kg)-24h group can reduce the level of serum D-lactic acid,but FTRAs has no obvious effect during and after the immune transition.(3)Administered before the immune transition,SAP+FTRAs(45mg/kg,90mg/kg)-24h group can reduce intestinal IL-1β,TNF-α,IL-6,IL-8,IL-18,TGF-β,IL-10 and plasma endotoxin content,increase the level of IL-4 and s TNF-αR.In the immune transition,the SAP+FTRAs(90mg/kg)-72h group can significantly reduce the levels of TNF-α,IL-18,and TGF-β.6.IL-8,ET,IL-10,IL-4,s TNF-αR have no obvious effect.After the immune transition,the SAP+FTRAs(90mg/kg)-96h group can significantly reduce IL-18,and the effect on IL-1β,TNF-α,IL-6,IL-8,ET,TGF-β,IL-10,IL-4 and s TNF-αR have no obvious effect.(4)The pathological changes of MLN found that the lymphatic follicles in the lymph nodes of the SAP+FTRAs(90mg/kg)-24h group were significantly reduced when administered before the immune transition,compared with the SAP-24h group.In the immune transition,compared with SAP-72h group,SAP+Rhubarb-72h lymph node pathological score decreased.After the immune transition,the drug has no obvious effect on the lymph nodes.Before the immune transition,compared with the SAP-24h group,the expression of CD68 and CD103 in the intestinal tract of the SAP-FTRAs(90mg/kg)-24h group was significantly increased.During the immune transition,it was found that compared with the SAP-72h group,the expression of CD103 in the intestinal tract of SAP+FTRAs(90mg/kg)-72h increased significantly,while the expression of CD68 did not change significantly.After the immune transition,compared with the SAP-96h group,the expression of CD68 and CD103 in the intestinal tract of the SAP+FTRAs treatment group did not change significantly.Before the immune transition,compared with the SAP-24h group,the SAP-FTRAs(45mg/kg,90mg/kg)-24h group can reduce the proportion of Th1,but has no effect on Th2,and the ratio of Th1/Th2 is reduced.In the immune transition,compared with the SAP-72h group,the SAP-FTRAs(45mg/kg,90mg/kg)-72h group can increase the proportion of Th2,but there is no significant change in the proportion of Th1,and the ratio of Th1/Th2 is reduced.small.After the immune transition,compared with the SAP-96h group,the ratio of Th1 and Th2 in the SAP-FTRAs-96h treatment group did not change significantly,and Th1/Th2remained basically unchanged.Before the immune transition,compared with the SAP-24h group,the SAP+Rhubarb-24h and SAP+FTRAs(45mg/kg,90mg/kg)-24h group can significantly increase the intestinal SIg A content.Compared with the SAP group,the SAP+FTRAs treatment group had no obvious effect on SIg A during and after the immune transition.(3)The protective effect of FTRAs on secondary infections in SAP rats:(1)The results showed that the 7-day mortality rate in SAP group and SAP+PA group were30%and 80%,respectively,while SAP+FTRAs(90mg/kg)+PA group 7 The daily mortality rate is 60%and the mortality rate is significantly reduced.(2)Compared with SAP group,TNF-αin SAP+PA group was significantly reduced,IL-1β,IL-6,IL-8,IL-18,plasma ET,TGF-β,IL-10,IL-4,s TNF-αR There is no significant change inαR.Compared with the SAP+PA group,the SAP+FTRAs(90mg/kg)+PA group has intestinal IL-1β,TNF-α,IL-6,IL-8,IL-18,plasma ET,TGF-β,IL-10.IL-4 and s TNF-αR have no obvious effect,but can reduce the level of IL-18.The SAP rats treated with FTRAs and secondary to PA infection can reduce the release of inflammatory cytokines caused by PA,which has a protective effect.Conclusion:(1)48h after modeling is the turning point of SAP rats from the period of excessive inflammatory response and mixed immunity to the period of compensatory immunosuppression.(2)FTRAs administered before the SAP immune transition can effectively inhibit the body’s inflammatory response,which is the best time for administration.(3)The administration of FTRAs before the immune transition can protect SAP from PA infection after the immune transition,and further clarify the best time of administration. |
PDF Full Text Request