| Part 1:Evaluation of maternal thyroid injury during pancreatitis in pregnancyObjective: To establish a model of severe acute pancreatitis(SAP)in pregnant rats and to observe thyroid injury during SAP in late pregnancy rats.Method: We randomly divided the purchased 36 experimental rats into sham-operated(SO)group,and combined severe acute pancreatitis of pregnancy(APIP)group according to the random number table method.Each group was subdivided into three subgroups of 3 h(APIP3h group),6 h(APIP6h group)and 12 h(APIP12h group),with 6 rats in each group.The APIP model was induced by retrograde homogeneous injection of 5% sodium taurocholate through the standard biliopancreatic duct and the bilateral biliopancreatic ducts were blocked for 5 min.Rats in each group were dissected and sampled according to different time points.The levels of amylase(AMY),lipase(LIP),free triiodothyronine(FT3)and free thyroxine(FT4)in the vein blood of the rats were measured by a fully automated biochemical analyzer,and the levels of TNF-α,IL-1β and IL-6 in the serum were measured by enzyme-linked immunosorbent,and the histological changes in the pancreas and thyroid were observed and scored microscopically.Results: Compared with SO group,serum AMY,LIP,TNF-α,IL-1β,IL-6 were significantly increased at different time points in APIP3 h,6h and 12 h groups,pancreas and thyroid tissue were damaged,and pathological scores were increased,and there were differences It is statistically significant(P<0.05);Compared between different time points,serum AMY,LIP,TNF-α,IL-1β and IL-6 levels and pancreatic and thyroid pathology scores were significantly higher in the APIP6 h and APIP12 h groups compared to the APIP3 h group,with statistically significant differences(P<0.05);while serum AMY,LIP,TNF-α,IL-1β and IL-6 levels and pancreatic and thyroid pathology scores were not significantly different in the APIP12 h group compared to the APIP6 h group.The levels of serum FT3 and FT4 were stable in the SO group,while the levels of serum FT3 and FT4 in the APIP3 h,6h and 12 h groups decreased to different degrees compared with the SO group,and the differences were statistically significant(P<0.05);In APIP rats at different time points,PIP6 h was significantly lower than APIP3 h FT3,and the difference was statistically significant(P<0.05);the FT3 level in the APIP6 h group was slightly lower than that in the APIP12 h group,and the difference was not statistically significant.the FT4 level in the APIP6 h group was lower than that in the APIP3 and APIP12 h groups,but the differences were not statistically significant.Conclusions: The results of this study suggest that rats with acute necrotizing pancreatitis in late pregnancy may be accompanied by thyroid structural damage and hypothyroidism,and the damage is more obvious 6 hours after modeling.Part 2:Dose of MIF inhibitor ISO-1 in pregnant rats with acute pancreatitisObjective: To investigate the effects of different doses of ISO-1 on pancreatic pathological injury,liver function and the expression of thyroid MIF in APIP rats.Methods: We randomly divided the 30 purchased experimental rats into sham-operated(SO)group and combined severe acute pancreatitis of pregnancy(APIP)group according to the random number table method;The APIP group was subdivided into a low-dose ISO-1 pretreatment group(APIPL,1.75 mg/kg),a medium-dose ISO-1 pretreatment group(APIPM,3.5 mg/kg)and a high-dose ISO-1 pretreatment group(APIPLH,7 mg/kg),with six animals in each group.The APIP model was induced by retrograde uniform injection of 5% sodium taurocholate through the standard biliopancreatic duct.They were killed after 6 hours of modeling.Then it was injected intraperitoneally 30 min before STC injection.Rats in each group were dissected and sampled according to different time points.The levels of alanine transaminase(ALT)and glutathione transaminase(AST)in tail vein blood of rats were measured by automatic biochemical analyzer;the levels of TNF-α,IL-1β and IL-6 in serum were measured by enzyme-linked immunosorbent assay;the expression of MIF in thyroid gland of rats was detected by immunohistochemistry;the histopathological changes of pancreas were observed and scored under light microscope.Results: On the basis of the first part,we examined the expression of MIF in thyroid tissue at different time points during acute pancreatitis in pregnant rats.We found that the expression of MIF in thyroid tissue was low in the SO group,while the expression levels of MIF in the thyroid gland of rats at each time point of APIP3 h,APIP6h and APIP12 h were increased to different degrees;Among them,the MIF expression in APIP6 h and APIP12 h groups was significantly higher than that in APIP3 h group,and the difference was statistically significant(P<0.05),while there was no significant difference in MIF expression in APIP6 h and APIP12 h groups.In this part of the experiment,animal models were constructed by selecting the 6h time point and administered with ISO-1 intervention.Compared with the APIP group,pancreatic alveolar hemorrhage,necrosis and inflammatory cell infiltration were significantly improved after pretreatment with different doses of ISO-1,and pathological scores,inflammatory factors such as TNF-α,IL-1β and IL-6 levels and thyroid MIF expression levels were significantly reduced,and the differences were statistically significant(P<0.05);among them,the APIPM and APIPH groups were better than the APIPL group in The difference was statistically significant(P<0.05),while there was no significant difference between the APIPM and APIPH groups.Serum transaminase ALT and AST levels were stable in the SO group.Compared with the SO group,serum ALT and AST levels were significantly higher in the APIP group.The differences were statistically significant(P<0.05);compared with the APIP group,the serum levels of ALT and AST decreased after different doses of ISO-1 treatment,and the differences were statistically significant(P<0.05);compared with APIPL,the serum levels of ALT and AST in the APIPM and APIPH groups were significantly lower,and the differences were statistically significant(P<0.05);while There was no statistically significant difference between the APIPM group and APIPH group compared with each other.Conclusion: 1.Intraperitoneal administration of the MIF inhibitor ISO-1 to rats with acute pancreatitis in late pregnancy reduced pancreatic,liver and kidney injury and significantly inhibited the expression of MIF in thyroid tissue without significant pharmacological liver and kidney injury;2.The medium dose group(3.5 mg/kg)is the safe and effective optimal dose for improving the injury in rats with acute pancreatitis in late pregnancy.Part 3:Protective effect and mechanism of ISO-1 on maternal thyroid injury in pancreatitis of pregnancyObjective: To investigate the protective mechanism of ISO-1 on thyroid injury in APIP rats.Method: We randomly divided the purchased 18 experimental rats into sham-operated(SO)group,acute pancreatitis in pregnancy(APIP)group and medium-dose ISO-1 intervention(APIPL)group,with 6 rats in each group.The induction of APIP model is the same as before.The ISO-1 group was injected intraperitoneally 30 min before STC injection at a dose of 3.5 mg/kg.Rats in each group were dissected and sampled 6h after modeling.The expression of ICAM-1,NF-k B and CD3 in rat thyroid was detected by immunohistochemistry;CD68 expression in rat thyroid was detected by immunohistochemical method;ROS expression level in thyroid tissue was detected by ROS fluorescence staining;histopathological changes in pancreas were observed and scored under light microscope.The ultrastructural changes of rat thyroid gland were observed by transmission electron microscopy imaging technique.serum MDA level of pregnant rats was detected by MDA assay kit.Serum free triiodothyronine(FT3)and free thyroxine(FT4)levels were measured by the kit.Results: We found that histopathological damage of thyroid gland was significantly increased in APIP group rats compared with SO group,while ISO-1 pretreatment alleviated thyroid pathological damage and increased serum FT3 and FT4 levels with statistically significant differences(P<0.05).Ultrastructural observation by electron microscopy revealed significant vacuoles,microvilli and perinuclear gaps in the thyroid follicular epithelium and a significant reduction in secretory vesicles in the APIP group compared with the SO group;expanded RER pools and rippled heterochromatic nuclei were detected in the follicular cells;and ISO-1 pretreatment attenuated the altered thyroid ultrastructural damage.After induction of APIP,the expression of CD68,CD3,NF-k B,ROS,ICAM-1 and serum levels of oxidative stress indicator MDA were significantly increased in the thyroid of pregnant rats,with statistically significant differences(P<0.05);whereas,after pretreatment with ISO-1,significant reductions in CD68,CD3,NF-k B,ROS,ICAM-1 and MDA levels were observed,with statistically significant(P<0.05).Conclusion: ISO-1 reduced inflammation and oxidative stress,attenuated inflammation and thyroid injury in APIP by inhibiting MIF and thereby suppressing inflammatory cell recruitment. |
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