| Research BackgroundFunctional dyspepsia(FD)is a common gastroduodenal disorder with clinical symptoms such as epigastric pain,postprandial fullness and bloating,epigastric burning and early satiety,etc.The global prevalence of FD is as high as 10%-30%,which has a serious impact on patients’ quality of life.The pathogenesis of FD is complex and closely related to "disorders of gut-brain interaction".The current Western medical treatment methods mainly include lifestyle modification,medication and psychotherapy.Although the symptoms are relieved to some extent,the efficacy often fails to meet the clinical needs.Chinese medicine has gradually received widespread attention both domestically and internationally due to its better clinical efficacy as well as safety,and more and more patients are seeking Chinese medicine treatment methods.The pathogenesis of FD is very complex.In recent years,with the further study of pathophysiological mechanism,more and more studies believe that the key part of the integration of pathophysiological mechanism of FD is in the duodenum.Stress or diet and other factors can cause low-grade duodenal inflammation and mucosal barrier damage,leading to gastrointestinal sensory and motor dysfunction,resulting in a series of dyspepsia symptoms in patients.Recently,the interaction between duodenal microbiota and host has attracted more and more attention from researchers.It may not only play an important role in the pathogenesis of FD,but also be an important way for Chinese medicine to take effect.The Xiexin Tiaoshu Decoction has the effect of relieving fullness and pain.Our group has confirmed the high efficiency and safety of this method in treating FD through RCT clinical studies,and through in vitro molecular experiments and metabolomics studies,it has been confirmed that this method can regulate the oxidative stress response in FD rats,and may have certain regulatory effects on intestinal flora.ObjectiveBased on previous research,we aim to observe the effects of Xiexin Tiaoshu Decoction on the duodenal mucosal barrier,low inflammation and gastric sensitivity,and gastric motility in FD rats,and further elucidate the effects of Xiexin Tiaoshu Decoction on the duodenal mucosal barrier function through VDR/NF-κB/MLCK signaling pathway,as well as duodenal microbiome and metabolism.Research Method1.Meta-analysis:PubMed、EMbase、The Cochrane Library、Web of Science、CBM、WanFang Data、CNKI and VIP databases were used to search for randomized,double-blind,placebo-controlled trials recruiting adults with FD treated with Chinese herbal compound.Oral herbal compounding alone for FD were included,and the quality of the included literature was evaluated using the Cochrane Risk Bias Assessment Tool,and the effect sizes of each index were combined using Review Manager V.5.4 software.2.Animal experiments:(1)FD rat models were prepared by iodoacetamide gavage and divided into normal group,model group,Xiexin Tiaoshu group and mosapride group.(2)The expression of tight junction protein occludin and inflammatory factor IL-6 in the duodenal tissue of FD rats were detected by Western blotting;the expression of Cathelicidin Antimicrobial Peptide(CAMP)in the duodenal tissue was detected by immunohistochemistry.(3)FD rat models were prepared by iodoacetamide gavage and divided into normal group,model group,Xiexin Tiaoshu group,mosapride group,vitamin D deficiency group and vitamin D deficiency+Xiexin Tiaoshu group.The expression of VDR,NF-κB p65,phosphorylated NF-κB p65,MLCK protein in the duodenal tissue of FD rats was detected by Western blotting;qPCR technique to detect VDR mRNA levels in duodenal tissues;the expression of 1.25(OH)2D3 in the serum was detected by Elisa;immunofluorescence detection of VDR expression;the expression of occludin and CYP27B1 in the duodenal tissue were detected by immunohistochemistry.(4)16SrRNA sequencing of intestinal contents flora,qualitative and quantitative metabolomic analysis of duodenal tissues based on LC-QTOF platform.Research results1.Results of Meta-analysis on the efficacy and safety of Chinese herbal compound in the treatment of functional dyspepsia:A total of 24 clinical trials were included,including 2939 patients.Meta-analysis showed that the overall remission rate of FD treated with TCM compounds was significantly higher than that of placebo(73.8%vs 46.2%)[RR=1.50,95%CI(1.29,1.76),P<0.00001].Among the single symptoms,TCM compounds were superior to the placebo in improving epigastric pain[MD=-0.86,95%CI(-1.20,-0.53),P<0.00001],epigastric burning sensation[MD=-0.39,95%CI(-0.65,-0.14),P=0.003],postprandial fullness discomfort[MD=-1.55,95%CI(-1.86,-1.24),P<0.00001],and early satiety symptoms[MD=-0.92,95%CI(-1.27,-0.57),P<0.00001].Compared with the placebo,TCM compounds treatment can obviously improve TCM syndrome in patients with FD[MD=-5.58,95%CI(-7.55,-3.61),P<0.00001],gastric emptying rate[MD=12.22,95%CI(8.90,15.55),P<0.00001],and helped to improve patients’ quality of life[MD=11.27,95%CI(0.10,22.43),P=0.05].No severe adverse events were reported between the two groups[RR=1.30,95%CI(0.89,1.90),P=0.17].2.The results of gastric sensitivity and gastric emptying in FD model rats by intervention with Xiexin Tiaoshu Decoction:(1)Gastric sensitivity:before the intervention,at a pressure of 20 mmHg:there was no significant difference in the rate of change of the root mean square of EMG of the trapezius of the rats in each group(all P>0.05).Under the pressure of 40 mmHg,60 mmHg and 80 mmHg:compared with the normal group,the rate of change of the root mean square of the EMG were significantly higher in the model group,the Xiexin Tiaoshu Decoction group and the mosapride group(P<0.01 or P<0.05),and there were no statistical difference in the rate of change of the root mean square of the EMG between the groups except the normal group(all P>0.05),which was comparable.After the intervention,at a pressure of 20 mmHg:there was no significant difference in the rate of change of the root mean square of EMG in each group(all P>0.05).Under the pressure of 40 mmHg,60 mmHg and 80 mmHg:the rate of change of the root mean square of EMG of the model group was significantly higher than that in the normal group(P<0.01 or P<0.05),and the rate of change of the root mean square of EMG of the rats decreased significantly after the intervention of theXiexin Tiaoshu Decoction(P<0.01 or P<0.05),and there was no statistical difference between the Xiexin Tiaoshu Decoction group and the normal group.The rate of change of EMG in the trapezius of rats was not significantly reduced after the intervention of mosapride(all P>0.05).(2)Gastric emptying:The gastric emptying rate of rats in the model group was reduced compared with the normal group(P<0.01).The gastric emptying rate of FD rats was significantly improved by the intervention of Xiexin Tiaoshu Decoction or mosapride(both P<0.01),and there was no significant difference in gastric emptying rate between the Xiexin Tiaoshu group and mosapride group(P<0.05).3.The results of Xiexin Tiaoshu Decoction intervention on duodenal tight junction protein,antibacterial peptide and inflammation in rats with FD model.(1)Comparison of occludin expression in rat duodenal tissue:WB results showed that cocludin protein expression was significantly decreased in the model group compared with the normal group(P<0.05),and significantly increased after the intervention of XiexinTiaoshu Decoction(P<0.05).(2)Comparison of CAMP expression in duodenal tissues of rats in each group:immunohistochemical staining showed that CAMP was positively expressed in duodenal intestinal epithelial cells of FD rats,mainly distributed in the cell pulp.Compared with the normal group,CAMP expression was significantly decreased in the model group(P<0.05);Compared with the model group,CAMP expression was significantly increased in the Xiexin Tiaoshu Decoction group(P<0.05),and the difference between the Xiexin Tiaoshu Decoction group and the normal group was not statistically significant(P>0.05).CAMP expression was not significantly increased in the mosapride group compared with the model group(P>0.05).(3)Comparison of IL-6 expression in the duodenal tissues of rats:compared with the normal group,the duodenal IL-6 protein expression in the model group was significantly increased(P<0.01)and showed low expression;compared with the model group,the duodenal IL-6 protein expression in the Xiexin Tiaoshu Decoction group and the mosapride group was significantly decreased(both P<0.001).4.Results of the effects of Xiexin Tiaoshu Decoction on VDR/NF-kB/MLCK signaling pathway in FD model rats.(1)Effects of Xiexin Tiaoshu Decoction on VDR,NF-κB p65,phosphorylated NF-κB p65 and MLCK protein expression in duodenal tissues of FD model rats:compared with the normal group,the duodenal VDR protein expression in the model group increased slightly(P>0.05);Compared with the model group,the duodenal VDR protein expression was significantly higher in the Xiexin Tiaoshu Decoction group(P<0.01),and there was no significant change in the mosapride group(P<0.05).The correlation between the duodenal VDR and duodenal occludin protein expression in the Xiexin Tiaoshu Decoction group was positive(P=0.003).Compared with the normal group,NF-κB p65,phosphorylated NF-κB p65 and MLCK protein expression were significantly increased in the model group(P<0.05 or P<0.01);Compared with the model group,NF-κB p65,phosphorylated NF-κB p65 and MLCK protein expression were significantly decreased in the Xiexin Tiaoshu Decoction group(P<0.05 or P<0.01),NF-κB p65,phosphorylated NF-κB p65 and MLCK protein were not significantly reduced in the Mosapride group(all P>0.05).(2)The effect of Xiexin Tiaoshu Decoction on VDRmRNA expression in the duodenal tissue of FD model rats:compared with the normal group,VDRmRNA expression in the model group was significantly higher(P<0.05),and VDRmRNA expression in the Xiexin Tiaoshu Decoction group was slightly higher(P>0.05);Compared with the model group,VDRmRNA expression in the Xiexin Tiaoshu Decoction and mosapride group was slightly lower,and the difference was not statistically significant(P>0.05).(3)The effects of Xiexin Tiaoshu Decoction on Serum 1,25(OH)2D3 and duodenal VDR,occlusin,CYP27B1 in Vitamin D Deficiency FD Rats:Comparison of serum 1,25(OH)2D3 levels in rats of each group:Compared with the normal group,serum 1,25(OH)2D3 levels in rats of the model group showed an upward trend,but there was no significant difference between the two groups(P>0.05);Compared with the model group,the expression of 1,25(OH)2D3 in the serum of FD rats in the vitamin D deficiency group decreased,approaching a statistically significant level(P=0.071);Compared with the model group,the serum 1,25(OH)2D3 level of FD rats in the Xiexin Tiaoshu Decoction group increased,but the difference had no statistically significant(P>0.05).The Mosapride group had no significant effect on the serum 1,25(OH)2D3 level of FD rats(P>0.05);After the intervention of Xiexin Tiaoshu Decoction,the serum 1,25(OH)2D3 level in FD rats with vitamin D deficiency increased,but the difference had no statistically significant(P>0.05).Comparison of VDR expression in duodenal tissues of rats in each group:immunofluorescence results showed that compared with the model group,the duodenal VDR expression was significantly higher in the XiexinTiaoshu Decoction group(P<0.01),and the duodenal VDR expression was significantly lower in the vitamin D deficiency group compared with the model group(P<0.05),and the duodenal VDR expression was not significantly increased after the intervention of Xiexin Tiaoshu Decoction in the presence of vitamin D deficiency(P>0.05).Comparison of the expression of occludin and CYP27B1 in the duodenum of rats in each group:Immunohistochemical results showed that occludin was positively expressed in the duodenal epithelial cells,mainly distributed in the membrane of the intestinal epithelial cells and the proximal cytoplasmic region within the membrane.CYP27B1 was positively expressed in duodenal intestinal epithelial cells,mainly distributed in the cell plasma.In the model group,there was a significant decrease in the expression of occludin and a increase in the expression of CYP27B1 in the duodenum of rats(P<0.05).Compared with the model group,the expression of occludin in the vitamin D deficiency group was significantly lower than that in the model group(P<0.05),the expression of CYP27B1 was higher than that in the model group(P>0.05).After the intervention of Xiexin Tiaoshu Decoction,the expression of occludin in the duodenum of FD rats was significantly increased(P<0.01)and the expression of CYP27B1 in the duodenum of FD rats was significantly decreased(P<0.01).The improvement effect of Xiexin Tiaoshu Decoction on the levels of occludin and CYP27B1 in FD rats with vitamin D deficiency were weakened.There was no significant difference between the mosapride group and the model group(P>0.05).5.Effects of Xiexin Tiaoshu Decoctiona on intestinal flora and its metabolism in FD model rats.(1)Comparison of duodenal microbiota of rats in each group:the OTU numbers of duodenal intestinal flora in the normal group,the model group,the Xiexin Tiaoshu Decoction,the vitamin D deficiency group and the vitamin D deficiency+Xiexin Tiaoshu Decoction group were 262,246,226,73 and 45 respectively.There was a trend of decrease in Shannon index in the model goup compared with the normal group(P>0.05);Compared with the model group,the Ace and Shannon index in vitamin D deficiency group were significantly decreased(P<0.05);There was a trend of increase in Ace and Shannon index after the intervention of Xiexin Tiaoshu Decoction in FD model rats and vitamin D deficiency FD model rats,but the difference was not statistically significant(P>0.05).The comparison of species abundance at the phylum level and species level of duodenal intestinal flora showed that the dominant flora of duodenal contents in these groups were all Firmicutes.There was no significant difference in the abundance of lactobacillus acidophilus,Lactobacillus johnsonii,Lactobacillus intestinalis and Akkermansia muciniphil among normal goup,model group and XiexinTiaoshu Decoction group,but the abundance of Lactobacillus intestinalis and Akkermansia muciniphil in model group were lower than normal group and Xiexin Tiaoshu Decoction group.Compared with the model group,the abundance of Lactobacillus intestinalis and Lactobacillus johnsonii were significantly decreased in vitamin D deficiency group and the vitamin D deficiency+Xiexin Tiaoshu Decoction group(P<0.01).Xiexin Tiaoshu Decoction could significantly improved the bundance of Lactobacillus intestinalis of model rats.At the condition of vitamin D deficiency,the improvement effect of Xiexin Tiaoshu Decoction on the abundance of Lactobacillus intestinalis was weakened.In this study,there was a significant positive correlation between the abundance of Lactobacillus intestinalis in the duodenal contents of rats and the expression of occludin and VDR in the duodenum(P<0.01).(2)Comparison of metabolites in the duodenal tissues of rats in each group:The results of PCoA analysis showed that the normal and model groups differed in metabolite profiles,while the normal and Xiexin Tiaoshu Decoction groups showed similarities.A total of 108 differential metabolites(DEMs)were identified in the normal and model groups,with 6 upregulated and 102 downregulated in the model group compared with the normal group(FC≥1,P≤0.05,VIP≥1).Similarly,237 differential metabolites were identified between the model group and the Xiexin Tiaoshu Decoction group,with 191 up-regulated and 26 down-regulated in the Xiexin Tiaoshu Decoction group compared with the model group(FC≥1,P≤0.05,VIP≥1).The total number of differential metabolites between the model group and the normal group,and between the Xiexin Tiaoshu Decoction group and the model group were 53.The levels of metabolites such as 7,8-dihydroneopterin(log2FC=-1.244,P=0.046),Caprylic acid(log2FC=-1.050,P=0.015)and Glycocholate(log2FC=-0.509,P=0.033)in duodenal tissues were significantly decreased in the model group compared with the normal group,and after the intervention of Xiexin Tiaoshu Decoction,the levels of metabolites such as 7,8-dihydroneopterin(log2FC=2.069,P=0.002),Caprylic acid(log2FC=1.329,P=0.011),and Taurochenodeoxycholate(log2FC=0.424,P=0.020)increased significantly.The KEGG database was used to annotate the differential metabolites,and the model group showed weaker pathways in methane metabolism,protein digestion and absorption,and cathelicidin antimicrobial peptide(CAMP)resistance compared with the normal group,while the first two pathways and the cathelicidin antimicrobial peptide(CAMP)resistance pathway tended to be enhanced to some extent after the intervention of Xiexin Tiaoshu Decoction compared with the model group.Research conclusions1.Xiexin Tiaoshu Decoction can improve the function of duodenal mucosal barrier and inflammation,reduce gastric sensitivity and improve gastric motility in FD rats.2.The VDR/NF-κB/MLCK pathway may be one of the ways that Xiexin Tiaoshu Decoction improves the duodenal mucosal barrier in FD rats.2.Xiexin Tiaoshu Decoction can regulate the duodenal intestinal flora and metabolism,which may be one of the mechanisms to improve the duodenal mucosal barrier in FD rats. |
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