Study On The Mechanism Of Regulation Of Intestinal Aquaporins By Heptamethoxyflavone The Dry Component Of “Fengyan Pieces Fructus Aurantii”

Objective Fructus Aurantii(FA)is a clinically used qi remedy with strong drying properties,and is one of Jiangxi’s native medicinal herbs."Fengyan pieces Fructus Aurantii,a concoction of Citrus aurantii shells,is a milder drying herb,and is a specialty of Jiangxi Zhanghang.In our previous study,we showed that 3,5,6,7,8,3’,4’-heptamethoxyflavone(HMF;polymethoxyflavone)is one of the main components of the dryness of Fengyan pieces Fructus Aurantii shell.In this study,normal C57BL/6 male mice,human small intestinal epithelial cells(FHs74lnt),human colon tissue cells(CCD018Co)and C57BL-6J AQP3 knockout mice were used to observe the effects of polymethoxyflavone on the body fluid and the expression of aquaporins(AQPs).The study investigated the effect of polymethoxyflavonoids on intestinal water channel proteins.Method1.Effect of HMF on fluid and intestinal water channel protein in normal miceNormal mice were used as research subjects to investigate the effects of HMF on body fluid and intestinal water channel proteins,using water intake,diet,faecal water content,small intestinal propulsion rate,pathological changes in small intestinal and colonic tissues,AQP3,AQP5,AQP7 and AQP11 contents and relative expression of AQP3,AQP5,AQP7 and AQP11 m RNA in small intestinal and colonic tissues,and AQP3,AQP5,AQP7 and AQP11 protein expression in small intestinal and colonic tissues as observation indexes.2.HMF on the expression of AQP3,AQP5,AQP7 and AQP11 m RNA and proteins in human small intestinal epithelial cells and human colon tissue cellsHuman small intestinal epithelial cells(FHs74lnt)and human colon histocytes(CCD018Co)were selected for the study.The relative expression of AQP3,AQP5,AQP7 and AQP11 m RNA genes and the expression of AQP3,AQP5,AQP7 and AQP11 proteins were used as reference indicators to finally investigate the effect of HMF with the expression of related AQPs proteins.3.The effect of HMF,on the water regulation function of water channel protein AQP3 knockout miceTaking into account the results of overall animal experiments and in vitro cellular assays of HMF and related intestinal AQPs,and combined with relevant literature,the CRISPR/Cas9 technology was used to edit the AQP3 gene to achieve systemic knockdown,combined with AQP3 m RNA gene expression and AQP3 protein expression as indicators to clarify the reliability of gene knockdown.Through the observation of tonicity indicators,we further reveal whether HMF achieves its effect on the body tonicity mainly through regulating AQP3.4.Study on the mechanism related to the regulation of intestinal water channel proteins by HMF Normal C57BL/6 male mice and C57BL-6JAQP3 knockout mice were used as the main study subjects,and aldosterone(ALD)and antidiuretic hormone(ADH)hormone levels,as well as AVPR1 and AVPR2,the upstream receptor genes of AQP3,were used as indicators to investigate the mechanism of HMF regulation of intestinal aquaporins.Results1.Effect of HMF on fluid and intestinal water channel protein in normal miceThe experimental groups were blank group(Control),Citrus aurantium group(ZQ),polymethoxyflavonoid high dose group(GHMF),polymethoxyflavonoid medium dose group(ZHMF)and polymethoxyflavonoid low dose group(DHMF).The results of the general observation indexes showed that the mice in each dosing group were mentally irritable,with fluffy skin hair and yellow colour compared with the blank group.There was no difference between the dietary intake of the groups(P>0.05).On the third and sixth days of administration,ZQ,ZHMF and GHMF increased the amount of water drunk by the mice compared with the blank group,which was statistically significant(p<0.05);on the ninth day of administration,the amount of water drunk by each group increased compared with the blank group,among which the difference between the GHMF group was statistically significant(p<0.05);on the twelfth day of administration,compared with the blank group,the amount of water drunk by GHMF,ZHMF and GHMF increased compared with the blank group.On the 12 th day of administration,the water consumption of GHMF,ZHMF and DHMF increased compared to the blank group,and the difference was statistically significant(p<0.01).In the faecal water content index,on day 3 of administration,the faecal water content decreased in each dosing group compared to the blank group,with no significant difference seen(p>0.05).At day 6,the faecal water content of GHMF increased and that of ZQ,DHMF and ZHMF decreased compared to the blank group,with significant differences(p<0.05).At day 12 of administration,the stool water content decreased in ZQ and increased in GHMF,ZHMF and DHMF compared to the blank group,but there was no difference and no statistical significance(p>0.05).The results of submandibular gland measurements showed that the weight of submandibular gland decreased in the administered group compared to the blank group to the extent of ZHMF<DHMF<GHMF,respectively,with statistically significant differences(P<0.05).The results of stomach and lung organ index assay showed that compared to the blank group,the organ index of ZQ group increased in weight and the organ weight of GHMF,ZHMF and DHMF groups decreased.The results of liver and kidney organ measurements showed that organ weights increased in the ZQ group compared to the blank group,and organ weights decreased in the GHMF,ZHMF and DHMF groups compared to the ZQ.The results of the small intestinal propulsion rate index showed that the small intestinal propulsion rates of GHMF,ZHMF,DHMF and ZQ were significantly reduced compared to the blank group,and there were differences with statistical significance(p<0.05)to the extent that G HMF < ZQ < DHMF < ZHMF.HE staining of small intestinal tissues showed no abnormalities in the blank group,while multiple ulcers were formed in ZQ,GHMF,ZHMF and DHMF,with damage invading the mucosal muscle layer and rupture appearing,and necrosis of intestinal glands was common at the ulcers,replacing them with proliferating connective tissue with a large number of lymphocyte infiltration.The results of colonic tissue examination in mice showed no significant abnormalities in the blank group,ZQ,DHMF and ZHMF,while GHMF showed ulceration,rupture of the mucosal muscle layer and lymphocytic infiltration.The results of immunohistochemistry of small intestine tissues showed that AQP3 immunohistochemistry showed an increase in expression in all the administered groups compared to the blank group,with a statistically significant difference in the GHMF group(P<0.05).AQP5 immunohistochemistry showed a decrease in expression in all the administered groups compared to the blank group,with a statistically significant difference(P<0.01),with a statistically significant difference in the degree of expression of DHMF<ZQ<GHMF<ZHMF.AQP7 immunohistochemistry showed an increase in expression in each of the administered groups compared to the blank group,with a statistically significant difference(P<0.001)in the expression of GHMF<DHMF<ZHMF<ZQ.The immunohistochemical results of colonic tissues showed that AQP3 immunohistochemical results showed increased expression in each administration group compared to the blank group,with differences in the DHMF group,which were statistically significant(p<0.05),and in the GHMF group,which were statistically significant(p<0.01).AQP5 immunohistochemical results showed increased expression in each administration group compared to the blank group,with differences in the DHMF,ZHMF,and GHMF groups,which were statistically significant(p<0.01).There were differences in the DHMF,ZHMF and GHMF groups with statistical significance(p<0.01).AQP7 immunohistochemical results showed increased expression in each administration group compared to the blank group,with differences in the ZQ group with statistical significance(p<0.05)and differences in the DHMF,ZHMF and GHMF groups with statistical significance(p<0.01).The relative expression of AQPs in small intestine tissues by qRT-PCR showed that in the relative expression of AQP3 m RNA,DHMF expression was significantly up-regulated compared to the blank group with statistical significance(p<0.01),GHMF expression was down-regulated with difference(p<0.05),and there was no significant difference in the rest of the groups(p>0.05).In the relative expression of AQP5 m RNA,compared to the blank group,the expression was down-regulated in all administered groups,and there was a difference in the ZQ group,which was statistically significant(p<0.05).In the relative expression of AQP7 m RNA,compared to the blank group,the relative expression was significantly up-regulated in the GHMF group,which was statistically significant(p<0.05).The relative expression of AQP11 m RNA was up-regulated in all the administered groups compared to the blank group,and the difference was statistically significant in the ZQ group compared to the blank group(p<0.05)and in the DHMF,ZHMF and GHMF groups(p<0.01).In the qRT-PCR colon tissue,the relative expression of AQP3 m RNA was down-regulated in all the administered groups compared to the blank group,with a statistically significant difference in the DHMF group(p<0.01)and a statistically significant difference in the ZHMF group(p<0.01).In the relative expression of AQP5 m RNA,compared with the blank group,the expression was up-regulated in all the administered groups,among which there were differences in the DHMF and GHMF groups with statistical significance(p<0.05),and there were significant differences in the ZHMF group with statistical significance(p<0.01).In the relative expression of AQP7 m RNA,compared with the blank group,ZQ expression was up-regulated but not statistically significant(p>0.05),while the rest of the groups expressed down-regulation,among which,there was a significant difference with statistical significance in the GHMF(p<0.05)and ZHMF groups(p<0.0001).in the relative expression of AQP11 m RNA,compared with the blank group,there was a statistically significant difference with statistical significance(p<0.01).in the relative expression of AQP11 m RNA,compared with the blank group,there was a statistically significant difference with statistical significance(p<0.01).In the expression of AQP11 m RNA,DHMF expression was significantly up-regulated compared to the blank group with statistical significance(p<0.01),while there was no significant difference in the rest of the groups(p>0.05).The results of western blot showed that the expression of ZHMF protein was increased in the AQP3 and AQP5 protein expression levels in the small intestine tissues compared to the blank group,and decreased in the rest of the groups.In the small intestine,the expression of ZQ,ZHMF and GHMF increased in AQP7 compared to the blank group,and in AQP11,the expression of ZQ and ZHMF increased and that of DHMF and GHMF decreased compared to the blank group.In colonic tissues,AQP3,AQP5,AQP7 and AQP11 protein expression levels were increased in each administration group compared to the blank group,to the extent that ZQ<DHMF<ZHMF<GHMF,respectively.2.HMF on the expression of AQP3,AQP5,AQP7 and AQP11 m RNA and proteins in human small intestinal epithelial cells and human colon tissue cellsThe relative expression of AQPs in small intestine tissues by qRT-PCR,as shown in Figure2-2,showed that the relative expression of AQP3 m RNA was down-regulated at 10ug/ml,20ug/m L and 40 ug/m L administration concentrations compared to the blank group,with statistical significance(P<0.05).The relative expression of AQP7 m RNA was up-regulated at 10 ug/m L and 40 ug/m L compared to the blank group,but the difference was not statistically significant(P> 0.05).The relative expression of AQPs in colon tissues by qRT-PCR,as shown in Figures 2-3,showed that the relative expression of AQP3 m RNA was down-regulated at 10ug/m L,20 ug/m L and 40 ug/m L administration concentrations compared to the blank group,with no statistically significant difference(P>0.05).AQP5 m RNA expression was down-regulated at 10 ug/m L and20 ug/m L compared to the blank group,with no statistically significant difference(P>0.05),and up-regulated at 40 ug/m L,with a statistically significant difference(P<0.05).AQP7 and AQP11 m RNA relative expression was up-regulated at 10 ug/m L compared to the blank group,with no statistically significant difference(P>0.05),expression was up-regulated at both 20 ug/m L and40 ug/m L dosing concentrations,with differences that were statistically significant(P<0.05).Western blot showed that in human small intestinal epithelial cells,AQP3,AQP5 and AQP11 protein expression was reduced in the HMF-administered group compared to the blank group,while AQP7 protein expression was increased in the HMF-administered group compared to the blank group.In human colonic epithelial cells,AQP3,AQP5,AQP7 and AQP11 m RNA and protein expression was increased in the HMF-administered group compared to the blank group.3.The effect of HMF on water regulation function in water channel protein AQP3 knockout miceThe results for general indicators of water consumption showed that in the wild type control group(Wild type,WT),water consumption increased on days 3,6,9 and 12 compared to the blank group,with a statistically significant difference(p<0.01).There was a statistically significant difference in ZQ water intake compared to the blank group(p<0.01).On days 9 and12,the ZQ group showed an increase in water consumption compared to the blank group,which was statistically significant(p<0.01),while no difference was seen in the HMF administration group compared to the blank group after days 3、6、9and 12 Day(p>0.05).The results of the eating volume index,in the WT and AQP3-KO groups,were significantly different and statistically significant(p<0.01)compared to the blank group,indicating that the AQP3 knockout did not have an effect on appetite and eating.The results of faecal water content,as shown in Figure 3-3 and Tables 3-5 and 3-6,show that in the WT group,on days 3 and 6 of dosing,faecal water content decreased in each dosing group compared to the blank group,with a statistically significant difference(p<0.01).On the9 th day of administration,the stool water content increased in all dosing groups compared to the blank group,with a significant difference(p<0.01).In the AQP3-KO group,there was no statistically significant difference in the stool water content of each dosing group compared to the blank group on days 3,6,9 and 12(p>0.05).The relative expression of AQP3 m RNA in small intestine and colon by qRT-PCR showed that the relative expression of AQP3-KO AQP3 m RNA was significantly down-regulated compared to WT.The results of western blot showed that AQP3-KO AQP3 protein expression was not significant in small intestine and colon tissues compared to WT.4.Study on the mechanism related to the regulation of intestinal water channel proteins by HMF4.1 Effect of HMF on ALD and ADH hormone levels and AVPR1 and AVPR2 gene receptor expression in normal mouse small intestine and colon tissuesThe results of ALD in normal mouse small intestine and colon tissues showed that in small intestine tissues,ZQ and HMF up-regulated the expression level of ALD compared to the blank group.In the colon tissues,ZQ up-regulated ALD expression levels and HMF down-regulated ALD expression levels compared to the blank group,with a trend between the groups,but no significant difference was seen and was not statistically significant(p>0.05).The small intestine and colon ADH results showed that in small intestine tissue,ZQ and HMF upregulated the expression levels of ADH compared to the blank group.In the colon tissues,ZQ up-regulated ADH expression levels and HMF down-regulated ADH expression levels compared to the blank group,no difference was seen and no statistical significance was found(p>0.05)The qRT-PCR AVPR1 and AVPR2 relative expression results,as shown in Figure 4-2,showed that in the small intestinal tissue AVPR1 expression,there was an increase in relative expression in the ZQ vs DHMF group compared to the blank group,with no difference and no statistical significance(p>0.05),and a decrease in relative expression in the ZHMF vs GHMF,with no difference and no statistical significance(p>0.05).In small intestinal tissue AVPR2 expression,relative expression was increased in the ZQ and DHMF groups compared to the blank group,with no difference and no statistical significance(p>0.05),and relative expression was decreased in the ZHMF and GHMF,with no difference and no statistical significance(p>0.05).In colonic tissue AVPR1 expression,there was no statistically significant increase in relative expression in the DHMF,ZHMF and GHMF groups compared to the blank group(p>0.05).In the small intestine tissue AVPR2 expression,there was no statistically significant increase in the relative expression in the DHMF,ZHMF and GHMF groups compared to the blank group(p>0.05).4.2 Effect of HMF on ALD and ADH hormone levels and AVPR1 and AVPR2 gene receptor expression in small intestine and colon tissues of AQP3 knockout miceThe ALD assay showed that in WT small intestine,ZQ was up-regulated and HMF was down-regulated compared to the blank group,while in AQP3-KO small intestine,ZQ was up-regulated and HMF was down-regulated compared to the blank group,while in WT colon,ZQ was up-regulated and HMF was down-regulated compared to the blank group.In AQP3-KO colon tissues,ZQ was up-regulated and HMF was down-regulated.The results of the ADH assay showed that in WT small intestine tissues,expression levels were up-regulated in all dosing groups compared to the blank group.The expression level of ZQ was up-regulated and that of HMF was down-regulated in AQP3-KO colon tissues compared with the blank group.The qRT-PCR results showed that the relative expression of AVPR1 and AVPR2 in the small intestine and colon tissues of WT and AQP3-KO mice was up-regulated in each dosing group compared to the blank group.Conclusion1.The overall animal test can conclude that HMF has dryness and plays a role in the index of body fluids such as drinking water and faecal water content,while it can significantly delay the small intestinal propulsion rate and cause histopathological damage to the small intestine and colon including local ulceration,mucosal muscle layer breakage and lymphocyte infiltration.At the same time,HMF can regulate the relative expression of AQP3,AQP5,AQP7 and AQP11 and protein expression in intestinal tissues.2.In vitro experiments on human small intestinal epithelial cells(FHs74lnt)and colonic tissue cells(CCD018Co)at different concentrations of HMF showed that 10ug/ml,20ug/ml and40 ug/ml were the optimal dosing concentrations,and revealed that AQP3 protein expression was superior to other AQPs family proteins and correlated with the dosing concentration.3.The combination of qRT-PCR and western blot techniques for AQP3 m RNA gene expression and AQP3 protein expression as reference indicators proved that the AQP3 gene had been knocked out.It was found that there were differences between WT and AQP3-KO mice in the fluid indicators drinking water and faecal water content.Therefore,it was preliminarily verified that HMF may affect the water reabsorption and secretion in intestinal tissues through the regulation of AQP3,which in turn affects the body’s fluids.4.By using small intestinal and colonic tissues of normal mice and AQP3 knockout mice as indicators of ADH and ALD,as well as upstream receptor genes such as AVPR1 and AVPR2,HMF was shown to affect water secretion and absorption by upregulating upstream receptor genes such as AVPR1 and AVPR2,promoting their binding to ALD and ADH,and influencing the opening and closing of AQP3 water channels.