| Severe acute pancreatitis(SAP)is the most common cause of acute abdominal disease hospital admissions.Intestinal injury is secondary to SAP and results in aggravation of the systemic inflammation response,which is account for the high mortality rate of SAP.NLRP3 inflammasome plays a key role in a variety of diseases.Recent studies have shown that NLRP3 inflammasome can trigger caspase-1 activation and stimulate the release of pro-inflammatory cytokines IL-1β and IL-18 in response to external exogenous infections or cell damage.Some drugs could inhibit NLRP3 inflammasome activation by an AhRdependent signaling pathway,and AhR acts as a negative regulator of the inflammasome activation.In addition,AhR is widely expressed throughout the intestinal epithelium,and the activation of AhR in epithelial cells may play an important role in the development of intestinal inflammation.What’s more,naringenin has significant anti-inflammatory effects and can effectively relieve inflammatory response in animal models of acute pancreatitis,but mechanisms of the anti-inflammatory effects are still unclear.Objective:1.Establishing mice model of SAP,and observing the effects of naringin’s treatment on intestinal and pancreatic injury of SAP mice;2.To explore the effect of naringin intervention on the inflammation degree of SAP mice;3.Exploring if the effect of naringin intervention on SAP mice worked through AhR/NLRP3 signaling pathway at the molecular biological level;4.The molecular docking technique and related experiments were used to explore whether AhR can be used as the target of naringin and its mechanism.Material and methods:1.15 healthy male C57BL/6 mice were randomly divided into the SAP group(n=12)and the normal saline group(n=3).Mice in the SAP group were intraperitoneally injected with cerulein and lipopolysaccharide,and were randomly divided into the SAP group,NAR group,CH223191 group and DEX group then.Bleed were collected from the eyeballs of mice 12 hours after modeling,then sacrificed all the mice and collected intestinal samples.The pathological changes of pancreas and intestinal mucosa were observed by H&E staining,The expression levels of IL-1β,TNF-α and IL-6 in serum of mice were detected by enzyme-linked immunosorbent assay(ELISA),the expression of NLRP3,AhR,IL-1β,TNF-α and IL-6 of mice in each group were detected by reverse transcription polymerase chain reaction(RT-PCR),immunohistochemistry,and Western Blot.Auto Dock Tools was used to predict the conformations of naringenin-AhR bindings and Py Mol 2.4 was used to visualized them.2.RAW264.7 cells were cultured in vitro,treated with LPS to simulate the inflammatory environment,and some cells transfected with si AhR to block the expression of AhR,and then treated with naringenin in the vitro model.The cells were divided into 6groups: control group,LPS group,LPS+NAR group,LPS+NAR+si AhR group,LPS+NAR+si NC group and LPS+NAR+CH223191 group.The cells were collected to extract cell RNA and protein after 12 hours treatment.The effective of transfection was detected by q RT-PCR and Western Blot.The optimal concentration of naringenin was screened by CCK8 assay.The expression levels of IL-1β,IL-6 and TNF-α in cells were detected by ELISA and the expression of NLRP3,AhR,IL-1β,IL-6 and TNF-α in the cells were detected by q RT-PCR and Western Blot.The nuclear translocation of AhR was detected by cell immunofluorescence(IF)and Western Blot.Results:1.Effects of naringenin intervention on abdominal organ injury in SAP mice:(1)There was no obvious edema,bleeding and necrosis in pancreas and intestines of the mice in NS group,NAR group and DEX group.While mice in SAP group and CH223191 group showed obvious necrosis and bleeding of pancreas and intestines with abdominal fluid by gross observation.(2)The H&E staining of pancreas and intestines in SAP model group and CH223191 group showed obvious focal necrotic area,lobular structure destruction,intestinal villi rupture,mucosal damage,inflammatory cell infiltration and bleeding.However,in NS group,NAR group and DEX group showed no obvious abnormality.Statistical analysis showed significant differences in pancreatic and intestinal pathological scores among these groups.2.Effects of naringenin intervention on intestinal inflammation and intestinal mucosal barrier in SAP mice:(1)Results of ELISA and q RT-PCR experiments showed that compared with NS group,the expressions of inflammatory cytokines including IL-1β,IL-6 and TNF-α were significantly increased in SAP group.The secretion of inflammatory factors in NAR group and DEX group were significantly reduced compared with SAP group.(2)In addition,q RT-PCR and Western Blot analysis showed that the expression of Occludin and ZO-1 in SAP group was reduced.Meanwhile naringenin inhibited the intestinal mucosal damage,and CH223191 could reverse the protective effect of naringenin on intestinal mucosa.3.Possible mechanism of naringenin’s intervention on SAP mice:(1)Immunohistochemical analysis,q RT-PCR and Western Blot analysis showed that the NLRP3 expression was significantly increased in SAP group compared with NS group.However,the expression of NLRP3 was decreased in the NAR group,similarly,the inhibitory effect of naringenin on NLRP3 activation was reversed after CH223191 treatment,and the expression of NLRP3 was significantly increased in CH223191 group,while relatively low in DEX group.(2)Auto Dock4.2.6 was used for molecular docking,and the binding energy between AhR and naringenin was-7.36 kcal/mol,showing significant binding activity.3D docking results showed that there were two hydrogen bonds between AhR and naringenin.Meanwhile,Western Blot demonstrated that naringenin treatment could promote the expression and nuclear translocation of AhR,while CH223191 could block the activation of AhR by naringenin.4.Establishment of the AhR knockdown cell model and effect of naringenin’s intervention on inflammatory response of RAW264.7 cells:(1)q RT-PCR and Western Blot results showed that the AhR knockdown cell model was successfully established.CCK8 experiment showed that 50μM naringin had a significant inhibitory effect on the proliferation of inflammatory cells.(2)ELISA results showed that the expressions of IL-1β,IL-6 and TNF-α were significantly up-regulated in LPS group compared with NC group and LPS+NAR group,suggesting that naringenin could reduce the expressions of IL-1β,IL-6 and TNF-α.In addition,the expressions of IL-1β,IL-6 and TNF-α in LPS+NAR+si AhR and LPS+NAR+CH223191 groups were higher than those in LPS+NAR+si NC group,suggesting that CH223191 and si AhR could reverse the anti-inflammatory effects of naringenin.5.Mechanism of naringenin’s intervention on activation of NLRP3 inflammasome in RAW264.7 cells:(1)Western blot and q RT-PCR analysis showed that NLRP3 expression was significantly up-regulated in RAW264.7 cells treated with LPS.The expression of NLRP3 was decreased after naringenin treatment.Suggesting that CH223191 and si AhR also reversed the effect of naringin.(2)IF and Western Blot showed that naringenin promoted the nuclear translocation of AhR in RAW26.7 cells and increased the expression of AhR,while CH223191 and si AhR inhibited the nuclear translocation of AhR.Conclusion:Naringenin mitigated SAP-related intestinal damage by reducing NLRP3 inflammasome activation,which is mediated partly by the AhR pathway.AhR may be a promising therapeutic target for naringenin in the treatment of SAP induced intestinal injury. |
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