Patchouli Alcohol Ameliorates DSS-induced Colitis By Activating PXR And Inhibiting NF-KB

ObjectiveInflammatory bowel disease is a group of chronic inflammatory diseases involving the gastrointestinal tract.Its main clinical manifestations are diarrhea,abdominal pain,mucus and bloody stools,which seriously affect people’s quality of life.It is now one of the five most common gastrointestinal diseases in the United States.However,the incidence has risen sharply in China and other Asian countries in recent years,and the current treatment of IBD has certain side effects,such as rifaximin liver and kidney.toxicity.Patchouli Alcohol is a commonly used aromatized wet drug.Some studies have reported its anti-inflammatory effect,but its anti-IBD mechanism has not been reported in detail,and further research is needed.At present,the role of PXR against IBD has been reported,and NF-ΚB and PXR have an interaction relationship.On this basis,this subject mainly uses molecular biological methods in vivo and in vitro to explore the anti-IBD effect of patchouli alcohol from PXR-NF-ΚB signaling pathway.Method1.Effects of patchouli alcohol on PXR and its target gene/protein expressionFirst,the hPXR-mediated CYP3A4 promoter activity was detected in HEK293T cells by luciferase assay,and the gene/protein expression of hPXR,CYP3A4 and MDR1 was detected by RT-PCR and Western Blot in LS174T cells.Immunofluorescence and UPLC-MS/MS were used to detect CYP3A4 expression and CYP3A4 enzyme activity,respectively.Then,mouse primary hepatocytes were used as cell model to detect gene expression of mPXR,Cyp3a11 and Ugtlal by RT-PCR,and Cyp3a11 protein expression was detected by immunofluorescence.Finally,Western Blot、immunofluorescence was used to detect the protein expression of hPXR in nucleus and cytoplasm of LS174T cells.2.The effect of patchouli alcoholon PXR and its target gene/protein expression after silencing PXRRT-PCR and WB assay were used to detect the gene and protein expression of hPXR and CYP3A4 in LS174T cells transfected with SiRNA-Control and SiRNA-PXR.UPLC-MS/MS was used to detect enzyme activity of CYP3A4 in LS174T cells treatment with PA after SiRNA-Control and SiRNA-PXR.3.Effects of patchouli alcohol on NF-K B and its downstream inflammatory factorsBased on THP-1 cells,LPS/TNF-α induced inflammation model,and the expression levels of NF-ΚB,IL-1β,IL-6,COX-2 and TNF-α were detected by RT-PCR and WB.The protein expression levels of NF-ΚB,p-NF-ΚB,IΚBα,and p-IΚBα protein were detected by WB and the nuclear and cytoplasmic proteins of NF-κB were detected by immunofluorescence and WB.4.Effects of patchouli alcohol on NF-K B and its downstream inflammatory factors after silencing PXRUsing LS174T cells as a model,luciferase assay was used to detect the activity of CYP3A4 promoter mediated by PXR.RT-PCR was used to detect the mRNA expression levels of NF-ΚB,IL-1β,COX-2 and ICAM-1.The total protein expression of NF-К B,p-NF-ΚB,IΚBα and p-IΚBα and the amount of NF-κB nuclear protein and plasma protein were detected by WB.After transfecting of SiRNA-Control and SiRNA-PXR,immunofluorescence were detected by the expression levels of NF-ΚB in the nucleus and cytoplasm of the cells.The mRNA levels of hPXR,NF-К B,IL-1β and IL-6 were detected by RT-PCR in the cells transfected with SiRNA-Control and SiRNA-PXR.5.Anti-inflammatory pharmacodynamics of patchouli alcohol on DSS-induced mice colitisC57BL/6 mice were administered by continuous intragastric administration for 9 days.On the third day,except for the blank control group,4%DSS was given.During the period,the body weight and bloody diarrhea of the mice were observed and recorded.After completing modeling and administration,open the mice abdominal cavity,cut the colon part,measure the length of the colon,make DAI score,HE staining to investigate the histopathological phenomenon of PA on DSS-induced colitis in mice,kit to detect content of I1-1 β,I1-6,I1-10,Tnf-α in colon tissue,RT-PCR detected gene expression of I1-1β,I1-6 in colon tissue,immunofluorescence detection of CD45,CD11b expression in tissues.6.Anti-inflammatory mechanism of patchouli alcohol on DSS-induced mice colitisThe expression of Cyp3a11 and Ugtlal in colon tissue was detected by RT-PCR.The expression of Cyp3a11 in colon was detected by immunofluorescence.The expression of NF-κB and mPXR in colon tissue was detected by fluorescence double staining.7.Anti-inflammatory effects of patchouli alcohol on DSS-induced colitis in mice after pharmacological inhibition of PXRThe mice were divided into the following groups:blank control group,KET group,DSS group,DSS+KET group,PCN+DSS group,PCN+DSS+KET group,PA+DSS group,PA+DSS+KET group.According to the above group,the mice were intragastrically administered with KET for 2 days,and then continuously administered for 10 days.Then,PCN and PA were intragastrically administered from the third day for 10 days,and the model mice were given 4%DSS(Distilled water dissolved)on the 5th day.while control mice were given distilled water for drinking.DAI scores were obtained according to mice body weight and bloody diarrhea.HE staining was used to detect the pathological changes of colon tissue.RT-PCR was used to detect the mRNA expression of mPXR,Cyp3a11,Ugtlal,Tnf-α and I1-6 in colon tissue.Results:1.Patchouli activates PXR signalMedium and high doses of PA up-regulated hPXR-mediated CYP3A4 promoter activity and CYP3A4 activity in LS174T cells.Medium and high doses of PA also increased CYP3A4 gene/protein expression,but had no effect on hPXR and MDR1 genes/proteins.,indicating that the PA can activate the hPXR-CYP3A4 signal.Considering that the LBD domain of PXR has significant species differences,we further investigated whether PA can activate mPXR signaling in mice.Similarly,we showed that medium or high doses of PA up-regulated the expression of Cyp3a11 and Ugtlal in mouse primary hepatocytes,but had no effect on the mPXR gene,suggesting that PA can also activate PXR signaling in mice.The positive control hPXR agonist(RIF)and the murine PXR agonist(PCN)also exerted a significant effect on the induction of CYP3A4/Cyp3a11.In addition,mechanistic studies have shown that different doses of PA promote the expression of hPXR into the nucleus and induce the expression of hPXR’s downstream target genes.Therefore,our results show that PA may be a PXR agnist that activates the PXR signal.2.After silencing PXR,patchouli alcohol lost the effect of activating hPXR signalingTo further explore the important role that hPXR plays in PA regulation of CYP3A4 induction,we performed hPXR knockdown experiments.After successful transfection of SiRNA-hPXR,PA and RIF reduced the up-regulation of CYP3A4 gene、protein and enzyme activity by PA/RIF.The results showed that the induction of CYP3A4 gene by PA was dependent on hPXR.Therefore,in summary,hPXR is indispensable for PA-mediated expression of CYP3A4.3.Patchouli alcohol inhibits NF-κB-regulated inflammatory signalsPXR activation has a significant anti-inflammatory effect and plays an important regulatory role in the anti-inflammatory treatment of IBD.To clarify whether PA has an anti-inflammatory effect,we first investigated whether NF-ΚB has an important inhibitory effect after PA treatment.LPS induction significantly increased the mRNA expression levels of NF-K B,IL-1β,IL-6,COX-2 and INF-α in THP-1 cells.However,different doses of PA interfered with cells and decreased LPS-induced mRNA expression levels of NFK B、IL-1β、IL-6、COX-2、TNF-α.Up-regulation of-1β,IL-6,COX-2,and TNF-αgenes;consistent with gene expression,different doses of PA reduced the phosphorylation levels of NF-κB and IκBα induced by LPS;in addition,different doses of PA could reverse LPS The function of the induced NF-κB protein into the nucleus.Therefore,we have shown that PA can significantly inhibit the inflammatory pathway that inhibits NF-κB regulation and exert an anti-inflammatory effect.4.Patchouli alcohol inhibits NF-κB regulation by activating hPXR signalingCrosstalk between PXR and NF-κB and inhibit the activity of NF-κB,which is an important mechanism for PXR to exert anti-inflammatory.Considering that PA is an agonist of PXR and that PA has a significant inhibitory effect on NF-κB signaling,we hypothesized that the anti-inflammatory effect of PA inhibiting NF-κB may be achieved by PXR activation.Therefore,we first conducted a PXR and NF-κB interaction study.After transfection of hPXR and NF-КB-luc plasmids,both medium and high doses of PA and RIF could down-regulate NF-K B-luc luciferase activity,suggesting that hPXR can inhibit NF-K B signaling.Considering the low expression of PXR in macrophages,we used LS174T cells(a PXR signal cell with strong endogenous expression)to further determine whether the inhibition of NF-κB by PA is activated by activating hPXR..We show that LPS induction can significantly increase NF-K B,IL-1β,COX-2,and ICAM-1 gene expression levels in LS174T cells.However,different doses of PA activate hPXR signaling in LS174T cells,which significantly reduces LPS induced NF-ΚB transcript levels and in turn lead to inhibition of NF-κB downstream target gene pro-inflammatory factors such as IL-1β,COX-2,ICAM-1 mRNA levels;consistent with transcription levels,different doses of PA and RIF activated hPXR also reduce TNF-α-induced phosphorylation of NF-κB and IκBα protein.Nuclear translocation of NF-K B is an important mechanism by which NF-κB regulates the expression of pro-inflammatory factors in downstream target genes.Therefore,preventing nuclear translocation of NF-КB plays a key role in inhibiting NF-κB-mediated inflammation.It was shown that PA significantly reversed the function of TNF-α-induced NF-K B protein transfer into the nucleus.The above results indicate that PA can significantly inhibit NF-κB regulation of inflammatory signals by activating PXR.5.After silencing PXR,patchouli alcohol reduces the inhibitory effect of NF-κB on the inflammatory pathwayTo further investigate the important regulatory role of hPXR in the inhibition of NF-κB regulation of inflammatory signaling by PA,we performed hPXR knockdown experiments.The results showed that PA could not reverse the TNF-α-induced NF-K B protein transfer into the nucleus after transfection of SiRNA-PXR,and lost the down-regulation of NF-κB,IL-1β and IL-6 genes induced by TNF-α.Therefore,after the loss of hPXR,PA attenuated the inhibition of NF-κB regulation of inflammatory pathways.The results indicate that PA inhibits NF-K B signaling and exerts anti-inflammatory effects that require hPXR involvement.6.Patchouli alcohol improves DSS-induced mouse colitisOur studies have reported that PA has anti-inflammatory effect.DSS induced mice colitis model was used in this experiment.DSS-induced mice showed severe clinical pathological symptoms of colitis:significantly decreased body weight on the 4th day、bloody diarrhea on the 6th day、shortened colon、DAI disease index increased and severely damaged colonic morphological structure.However,after treatment with PA or positive drug RIF,the clinical symptoms of these colitis induced by DSS can be significantly improved;further detection of the expression of inflammatory factors in colon tissue After treatment with PA or positive drug PCN,the content of Il-1β,Il-6,Il-10,Tnf-α and the mRNA expression of Il-1β and Il-6 in DSS-induced colonic tissues of mice were significantly decreased.It was shown by immunofluorescence that the protein expression levels of CD45 and CD11b were decreased after PA or PCN treatment.The above results show that PA has a significant effect on reducing DSS-induced inflammation and improving DSS-induced inflammatory bowel disease.7.Patchouli alcohol regulates PXR/NF-КB signal in mice colon induced by DSSTo elucidate the in vivo mechanism by which PA activates mPXR and inhibits NF-κB signaling,we determined the effect of PA on mPXR and NF-κB signaling in mice colon tissues.Firstly,the expression of mPXR target gene-Cyp3a11 and Ugtlal genes in mice colon tissues was determined.The results showed that simple administration of PA or PCN could significantly increase the mRNA expression of Cyp3a11 and Ugtlal in mice colon tissues;further immunofluorescence showed that PA or PCN increased the protein expression of Cyp3a11 in mice colon tissue induced by DSS.Moreover,the results of fluorescent double staining showed that PA or PCN inhibited DSS-induced protein expression of NF-κB and promoted mPXR protein in mice colon tissue.These results indicate that PA activates mPXR and inhibits the NF-κB signaling pathway.8.Pharmacological inhibition of PXR reduces patchouli alcohol to improve DSS-induced colitis in miceWe inhibited the transmission of PXR signaling by pharmacological inhibition with KET(PXR antagonist),and further explore whether PA can alleviate the protective effect on DSS-induced colitis.The results showed that mice induced by DSS increased susceptibility to colitis:weight loss,severe diarrhea,colon shortening,elevated DAI index,colonic histopathological damage,and extensive inflammatory cell infiltration.However,the above clinical symptoms were attenuated after PCN or PA administration.while the pretreatment of PXR pharmacological inhibitors-KET in mice,PA can not alleviate the clinical manifestations of colitis.Further,we explore the regulation of PA on mPXR target genes after mPXR inhibition,the results show that pretreatment Inhibition of mPXR-KET,PA can not up-regulate the mRNA expression of Cyp3a11,Ugtlal in mice colon tissue.PA inhibits mPXR signaling pathway.Finally,we measured the level of inflammatory factor gene in colon tissue,the results indicates that pretreatment of KET attenuated the effect of PA on inhibiting Tnf-α and I1-6 mRNA.Conclusion:PA is an agonist of PXR,which has anti-inflammatory effects on DSS-induced mouse colitis,and its anti-colitis mechanism is achieved by activating PXR and inhibiting the NF-κB signaling pathway.