| Objective: Acute lung injury(ALI)is one of the most common extra-pancreatic organ injuries in patients with severe acute pancreatitis(SAP)and is closely associated with high mortality in SAP patients.Alveolar macrophages(AMs)are important players in the pulmonary immune response and are highly plastic,polarizing into different functional phenotypes in response to different stimuli,and the transformation of AMs into functional phenotypes plays an important role in the disease regression process.The stimulator of interferon genes(STING)signalling pathway,which senses the abnormal presence of DNA in the cytoplasm,induces type I interferon production and enhances inflammatory cytokine production,has been shown to play an axial role in infectious and autoimmune diseases,but it has been shown to play an important role in acute lung injury associated with severe acute pancreatitis(SAP-associated acute lung injury).However,its specific mechanism of action in SAP-associated acute lung injury(SAP-ALI)remains to be fully explored.Emodin(EMO)is derived from rhubarb and is one of the effective monomeric components of qingyi decoction(QYD),a formula commonly used in clinical treatment of SAP.In this experiment,a rat SAP-ALI model induced by retrograde injection of 5% sodium taurocholate(STC)into the biliopancreatic duct was used to investigate the role of STING signalling pathway in its pathogenesis and its effect on the polarisation of AMs,and to compare the effects of STING-specific inhibitor C-176,STING-specific agonist DMXAA and This study will further explore the mechanism of action and potential targets of rhodopsin in the treatment of SAP-ALI,and provide new treatment ideas for the prevention and treatment of SAP-ALI patients in clinical settings.Method:In this experiment,the SAP-ALI model was constructed by retrograde injection of 5% STC into the pancreaticobiliary duct.Fifty 6-to 8-week-old Sprague-Dawley(SD)male rats were fed ad libitum for 7 days and their body weight was maintained at 220±20g.Then 50 SD rats were randomly divided into 5 groups : control group(Sham operation,SO),disease model group(SAP),C-176(STING inhibitor)intervention group(C-176),emodin intervention group(EMO),DMXAA(STING agonist)+emodin intervention group(DMX+EMO),each group contained 10 rats.The rats in the SAP group were injected with 5% STC(50mg/kg)retrograde into the pancreaticobiliary duct to establish the SAP rat model,while the SO group only turned the pancreas and surrounding tissues a few times after opening the abdomen.C-176 group was given C-176(10.5mg/kg)by intraperitoneal injection immediately after modeling;emodin(40mg/kg)was gavaged 30 min and 12 h after operation to EMO group;in DMX+EMO group,DMXAA(7mg/kg)was injected into the abdominal cavity immediately after operation,and then emodin was gavaged twice 30 minutes and 12 hours after operation.Samples in each group were collected 24 hours after modeling for the following experiments:(1)Detect the amylase activity in serum with the commercial kit.(2)Collect pancreatic tissue and some lung tissue,observe pathological sections of pancreatic and lung tissue and perform histopathological scoring.(3)Commercial ELISA kits were used to determine the levels of inflammatory factors IL-1β and TNF-α in rat serum and IL-1β and IL-10 in bronchoalveolar lavage fluid,respectively.(4)Check the genetic condition of STING,CD86,CD206 in rat lungs by q RTPCR.(5)Detect the protein of STING、IRF3、p-IRF3 in rat lungs by WB.(6)IF staining was used to detect the expression of CD86 and STING,markers of macrophage polarization in lung tissue.Results:(1)Compared with the SO group,the pancreatic and lung tissues of the rats in the SAP group showed significant pathological damage,with significantly higher pathological scores(p<0.001);meanwhile,amylase,IL-1β and TNF-α in the serum of the rats in the SAP group were significantly increased(p<0.001),and the W/D weight ratio of the lung tissues was increased(p<0.001);the protein content in BALF,IL-1β levels were also significantly increased compared with the SAP group(p<0.001,p<0.05),indicating that the SAP model was successfully constructed in this experiment.(2)By observing the results of WB,q RT-PCR and immunofluorescence,it was found that the expression of STING in the lung tissues of rats in the SAP group was significantly higher compared with the SO group(p<0.001,p<0.01),while the WB results showed that the phosphorylation level of IRF3 in the lung tissues of rats in the SAP group was increased(p<0.05),indicating that the STING signaling pathway in SAP was effectively The results of WB also showed that the phosphorylation level of IRF3 was increased in the lung tissue of the SAP group(p<0.05),indicating that the STING signaling pathway was effectively activated in SAP and aggravated the disease severity.Compared with the SAP group,WB,q RT-PCR and immunofluorescence results showed that the expression of STING was reduced in the lung tissues of the C-176 group(p<0.05,p<0.001)and the phosphorylation of IRF3 was reduced(p<0.05);the pathology of the pancreas and lung tissues of the C-176 group was improved(p<0.001),and the serum levels of amylase,IL-1β and TNF-α levels decreased(p<0.001),the wet/dry weight ratio of lung tissue decreased(p<0.01),and the protein content of BALF decreased(p<0.001),indicating that the inhibitor C-176 could effectively inhibit the activation of STING signaling pathway and alleviate the severity of the disease in rats.(3)Compared with the SO group,IL-1β secretion was increased in the BALF of rats in the SAP group(p<0.05),and IL-1β secretion was significantly reduced and IL-10 secretion was increased in the C-176 group(p<0.001).Meanwhile q RT-PCR results showed increased CD86 expression in SAP group(p<0.001)and significantly decreased CD206 expression(p<0.05).Immunofluorescence showed increased CD86 expression in SAP group and decreased CD86 expression in C-176 group,indicating that STING signaling pathway promoted M1-type polarization and inhibited M2-type polarization of AMs.(4)Compared with the SAP group,pancreatic and lung histopathology were improved in the EMO group(p<0.001),serum levels of amylase,IL-1β and TNF-α decreased(p<0.001),wet/dry weight ratio of lung tissue decreased(p<0.001),and protein content in BALF decreased(p<0.001);WB,q RT-PCR,immunofluorescence The results showed a decrease in STING expression(p<0.05,p<0.001)and IRF3 phosphorylation(p<0.05)in the lung tissues of rats in the EMO group.Compared with the EMO group,the DMX+EMO group showed increased histopathological damage to the pancreas and lung(p<0.01),increased serum levels of amylase,IL-1β and TNF-α(p<0.001,p<0.01,p<0.05)and increased wet/dry weight ratio of lung tissue(p<0.05);increased STING expression and IRF3 phosphorylation(p< 0.01,p<0.05),indicating that EMO could effectively inhibit STING signaling pathway activation to alleviate disease severity.(5)Compared with the SAP group,the secretion of IL-1β was decreased(p<0.05)and IL-10 was increased(p<0.001)in the BALF of rats in the EMO group.q RT-PCR showed a decrease in CD86 expression(p<0.001)and an increase in CD206 expression,and immunofluorescence showed a decrease in CD86 expression;compared with the EMO group,the secretion of IL-1β was increased(p<0.05)and IL-10 was increased(p<0.001)in the DMX+EMO group.IL-1β secretion was increased(p<0.05),IL-10 secretion was decreased(p<0.001),q RT-PCR results showed increased CD86 expression(p<0.001),decreased CD206 expression(p<0.05),and immunofluorescence showed increased CD86 expression,indicating that EMO promotes M2-type polarization of AMs.Conclusion:(1)STING signaling pathway is effectively activated in the lung tissue of SAP-ALI rats and plays a role in promoting inflammatory response;C-176 can effectively inhibit the activation of STING signaling pathway in vivo and alleviate the inflammatory response of the disease.(2)STING signaling pathway plays a pro-inflammatory role in SAP-ALI by changing the polarization direction of AMs,promoting M1-type polarization and inhibiting M2-type polarization.(3)EMO can play a pharmacological role in the treatment of SAP-ALI by inhibiting the activation of STING signaling pathway in the lung tissue of SAP-ALI rats and promoting the M2-type polarization of AMs. |
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