| ObjectiveTo establish a rat model of chronic restraint stress-induced diarrhea-predominant irritable bowel syndrome(IBS-D);to explore the regulation of muscularis macrophages(MMs)on the colonic neuronal homeostasis and intestinal smooth muscle function in IBS-D rats;and to discover the underlying mechanism of patchouli alcohol(PA)on the improvement of intestinal neuronal homeostasis and intestinal smooth muscle function in IBS-D rats based on the regulation of MMs on the intestinal smooth muscle function in IBS-D rats.Methods1.The effect of temporary depletion of MMs on the smooth muscle function of the intestine in IBS-D rats(1)Establishment and evaluation of IBS-D rat modelAn IBS-D rat model was established using an improved single-factor chronic restraint stress method,and the IBS-D rat model was evaluated by fecal areas,AWR score,charcoal transit rate in small intestine and isolated colonic smooth muscle tension.(2)The clearance efficiency of Pexidartinib on colonic MMs in IBS-D ratsAfter oral administration of the CSF1R inhibitor Pexidartinib,the proportion of CD11b/c+MHC Ⅱ+MMs to CD45+white blood cells in the muscular tissue of IBS-D rats were detected by flow cytometry.The number and distribution of CX3CR1+MMs were observed by full-layer slicing and laser confocal microscopy.qPCR was used to detect the mRNA expression level of CX3CR1 in the colonic tissue of IBS-D rats after temporary depletion of MMs.(3)The effect of temporary MMs depletion on intestinal smooth muscle function in IBS-D ratsThe changes of intestinal function in IBS-D rats with temporary MMs depletion were evaluated by measuring the feces area,fecal water content and AWR scores in small intestine.The changes of tension were detected by evaluating the autonomic contraction,electrical stimulation,addition of carbachol and high concentration KCl(120 mM),and gradient stretching(0.5-3.5 mm)in isolated colonic smooth muscle tension of IBS-D rats with temporary MMs depletion.(4)The effect of temporary MMs depletion on neuronal homeostasis in colonic longitudinal muscle myenteric plexus(LMMP)of IBS-D ratsThe change in the proportion of ChAT+neurons and nNOS+neurons in LMMP was detected by full-layer slicing and laser confocal microscopy after temporary MMs depletion.(5)The impact of temporary MMs depletion on the BMP/SMAD pathway in IBS-D ratsThe changes of colonic smooth muscle tension in IBS-D rats after the addition of BMP receptor inhibitor Dorsomorphin were observed.The changes of contraction tension in isolated colonic smooth muscle of IBS-D rats with temporary MMs depletion were observed after gradient addition of exogenous BMP2.The change in the number of pSMAD 1/5/8 and the proportion of pSMAD 1/5/8 to BMPR2 neurons in LMMP was detected by full-layer slicing and laser confocal microscopy in IBS-D rats with temporary MMs depletion.The mRNA and protein expression levels of BMPR2 and pSMADl/5/8 in colonic tissues of IBS-D rats with temporary depletion of MMs were detected by applying qPCR and western blot techniques.2.Study on the regulation mechanism of patchouli alcohol(PA)on colonic smooth muscle function dependent on MMs in IBS-D rats(1)The regulatory effect of PA on MMs in the colonic muscle layer of IBS-D rats with temporary MMs depletionAfter PA intervention,flow cytometry was used to detect changes in the proportion of CD 11 b/c+MHC II+MMs among CD45+white blood cells in the colon muscle layer of IBS-D rats with temporary MMs depletion.The number of CX3CR1+MMs were observed through full-layer slicing and laser confocal techniques.qPCR technology was used to detect the mRNA expression level of CX3CR1 in colon tissues.(2)The effect of PA on colonic smooth muscle function in IBS-D rats with temporary MMs depletionThe effect of PA on intestinal function was evaluated by detecting the fecal areas,fecal water content and AWR scores in small intestine.The regulation effect of PA on isolated colonic smooth muscle tension in IBS-D rats with temporary MMs depletion was also evaluated through colonic smooth muscle tension experiment in vitro.(3)The regulation of PA on neuronal homeostasis in the colonic LMMP of IBS-D rats with temporary MMs depletionFull-layer slicing and laser confocal techniques were used to observe changes in the proportion of colonic neuronal subtypes in the colonic LMMP of IBS-D rats with temporary MMs depletion under PA intervention.(4)The regulation of PA on the BMP/SMAD pathway in IBS-D rats with temporary MMs depletionBy using whole-layer slicing and laser confocal techniques,we detected the changes in the number of pSMAD 1/5/8,and the proportion of pSMAD 1/5/8 to BMPR2 neurons in the colonic LMMP of IBS-D rats with temporary depletion of MMs after PA intervention.The qPCR and western blot techniques were applied to detect the mRNA and protein expression levels of BMPR2 and pSMAD 1/5/8 in the colonic tissues of IBS-D rats with temporary depletion of MMs after PA intervention.Results1.The impact of temporary depletion of MMs on the intestinal smooth muscle function of IBS-D rats(1)Establishment and evaluation of IBS-D rat modelCompared with the control group,the feces area of IBS-D model rats on D14,D21,and D28 significantly increased.The residual water volume in the colonic distension experiment on D14 and D28 increased,and the charcoal transit rate in the small intestine on D28 increased.The results of isolated colonic muscle tension showed that the isolated colonic muscle tension of IBS-D rats was significantly higher than that of the control group at 0.5-3.5 mm stretching.These results suggested that the colonic motility was increased and the viscera sensitivity was heighted in IBS-D model rats,which indicated that the IBS-D rat model was successfully established.(2)The clearance efficiency of Pexidartinib on the colonic MMs of IBS-D ratsAfter oral administration of CSF1R inhibitor Pexidartinib,compared with the IBSD group,the proportion of CD11b/c+MHC Ⅱ+MMs in the colonic muscle layer of IBSD rats significantly decreased,and the number of CX3CR1+MMs in the colonic LMMP of IBS-D rats significantly decreased as shown by immunofluorescence results.The qPCR results showed that the mRNA expression of CX3CR1 in the colonic tissue of IBS-D rats after Pexidartinib intervention showed a synchronous decreasing trend compared with the IBS-D group.(3)Effects of MMs temporary depletion on intestinal smooth muscle function in IBSD ratsThe in vivo experiment results showed that,compared with the IBS-D group,the fecal water content of IBS-D rats with temporary MMs depletion increased significantly,the remaining water content at AWR score=3 was also significantly increased,and the feces areas showed an increasing trend.These results suggested that temporary MMs depletion caused further dysregulation of intestinal motility and visceral sensitivity in IBS-D rat.The experiment results in vitro showed that the tension of colonic smooth muscle of IBS-D rats with temporary MMs depletion exhibited an increasing trend compared to IBS-D rats under spontaneous contraction,KCl,carbachol or electrical field stimulation.The gradient stretching results of colonic smooth muscle strips showed that the tension of colonic smooth muscle strips in vitro of IBS-D rats with temporary MMs depletion increased significantly compared to IBS-D rats under stretching of 3.5mm.(4)Effects of MMs temporary depletion on neuronal homeostasis in colonic LMMP of IBS-D ratsImmunofluorescence results showed that the proportion of ChAT+neurons to Hu C/D+neurons showed an increasing trend compared to IBS-D rats.In addition,the proportion of nNOS+neurons to Hu C/D+neurons in IBS-D rats with temporary MMs depletion were similar to those in IBS-D rats.(5)The effects of MMs temporary depletion on BMP/SMAD pathway in IBS-D ratsCompared with IBS-D group,after adding the BMP receptor inhibitor Dorsomorphin,the isolated colonic smooth muscle tension of IBS-D rats showed a certain increase trend with a stretch of 2mm-3.5mm;compared with the control solvent(Kreb’s solution),after gradually adding exogenous BMP2(0.625,3.125,6.25 ng),the isolated colonic smooth muscle tension of IBS-D rats with MMs temporary depletion showed a significant decrease.In addition,compared with IBS-D rats,the number of pSMAD1/5/8+neurons in colonic LMMP of IBS-D rats with MMs temporary depletion significantly decreased,and the proportion of pSMAD1/5/8+to BMPR2+neurons also showed a significant decrease.The gene expression levels of pSMAD 1/5/8 in the colonic tissues of IBS-D rats with temporary depletion of MMs showed an overall decreasing trend,while the protein expression levels of pSMAD1/5/8 were significantly decreased compared to the IBS-D group.2.Study on the regulation of patchouli alcohol(PA)on the colonic smooth muscle function dependent on MMs in IBS-D rats(1)The regulation of PA on MMs-dependent colonic muscularis macrophages in IBSD rats with MMs temporary depletionAfter oral administration of low-dose(10 mg/kg)and high-dose(20 mg/kg)PA for 1 week,compared with the IB S-D+Pex group,the proportion of CD 11b/c+MHC Ⅱ+MMs in the colonic muscle layer of IBS-D rats with MMs temporary depletion was restored,and the high-dose PA had a significantly effect on restoring MMs;according to the results of immunofluorescence,compared with the IBS-D+Pex group,after lowdose and high-dose PA intervention,the number of CX3CR1+ MMs was also restored,and the expression level of CX3CRl mRNA showed an increasing trend,and the highdose PA could significantly increase the mRNA expression of CX3CR1.(2)The impact of patchouli alcohol(PA)on intestinal smooth muscle function in IBSD rats with temporary depletion of MMsIBS-D rats with MMs temporary depletion that received low-dose or high-dose PA interventions showed significant improvements in several aspects compared to the IBSD+Pex group,including fecal areas,AWR score and fecal water content in small intestine.In particular,the fecal area decreased,the remaining water volume at AWR score=3 reduced and the fecal water content declined under PA interventions.These results indicated that PA effectively improved the intestinal motility and visceral sensitivity in IBS-D rats with temporary depletion of MMs.In vitro results indicated that,compared with the IBS-D+Pex group,high-dose PA had a significant inhibitory effect on the high contraction state of isolated colonic smooth muscle in IBS-D rats with temporary depletion of MMs under spontaneous contraction,carbachol or electric field stimulation.Additionally,low-dose and highdose PA effectively reduced the muscle tension of the isolated colonic muscle strips in IBS-D rats with temporary depletion of MMs under gradient stretching compared to the IBS-D+Pex group.(3)The regulation of patchouli alcohol(PA)on the intestinal neuronal homeostasis in colonic LMMP of IBS-D rats with temporary depletion of MMsThe immunofluorescence results showed that,after low-dose and high-dose PA intervention the proportion of ChAT+neurons significantly decreased in IBS-D rats with temporary depletion of MMs compared with the IBS-D+Pex group.On the contrary,the proportion of nNOS+neurons significantly increased,and the proportion of intestinal neuronal subtypes tend to become stable.(4)The regulation of the BMP/SMAD pathway in IBS-D rats with temporary depletion of MMs by patchouli alcohol(PA)The immunofluorescence results showed that,after PA intervention,the number of pSMADl/5/8+neurons and proportion of pSMAD1/5/8+neurons to BMPR2+neurons significantly increased compared to the IBS-D+Pex group.In addition,compared with the IBS-D+Pex group,high doses of PA effectively increased the protein expression level of pSMAD1/5/8.,and the nuclear translocation function of pSMAD1/5/8 converged to normal levels.Conclusion1.Single-factor chronic restraint stress-induced IBS-D model rats showed increased intestinal motility,increased visceral sensitivity,and higher tension of isolated colonic smooth muscle,which is a stable,reliable,and ideal model for IBS-D.2.IBS-D rats with temporary depletion of MMs showed further increased intestinal motility,further heightened visceral sensitivity,and high contraction of isolated colonic smooth muscle tension,which was consistent with the result of increased intestinal motility in vivo.The results indicated that MMs had a significant regulatory effect on colonic smooth muscle function in IBS-D rats.Meanwhile,after temporary depletion of MMs,the proportion of excitatory motor neurons in colonic LMMP of IBS-D rats were more chaotic and fluctuating.In the BMP/SMAD pathway;the nucleoplasmic translocation function of pSMAD1/5/8 was significantly altered.The results indicated that MMs may regulate the nucleoplasmic translocation function of pSMAD1/5/8 through the BMP/SMAD pathway,affecting enteric neuronal homeostasis,and regulating colonic smooth muscle function in IBS-D rats.3.Patchouli alcohol has a desired recovery effect on MMs.Patchouli alcohol was effective in restoring MMs along with improving visceral hypersensitivity and intestinal motility and inhibiting the hypercontractile state of isolated colonic smooth muscle in IBS-D rats with temporary depletion of MMs.The recovery of MMs by patchouli alcohol also promoted the homeostatic balance of excitatory and inhibitory motor neurons in IBS-D rats with temporary depletion of MMs,while effectively restoring the number,proportion and nuclear translocation function of pSMAD 1/5/8 in the BMP/SMAD pathway.The results indicated that patchouli alcohol(PA)effectively restored MMs,regulated the nucleoplasmic translocation function of pSMAD 1/5/8 in BMP/SMAD pathway,maintained enteric neuronal homeostasis,improved intestinal smooth muscle function,and played a crucial role in treating IBS-D. |
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