| BackgroundSpleen-deficiency syndromeis one of the common TCM(traditional Chinese medicine)syndrome types in various intestinal diseases,with abdominal distension,abdominal pain,loose stool and so on.Intestinal mucosal injury is a common pathological manifestation in spleen-deficiency syndromeand many intestinal diseases,including shortening and loss of intestinal villi,irregular morphology of intestinal epithelial cells(IEC),and lamina propria edema.Mucosal repair after intestinal injury is an important way to restore and maintain intestinal barrier function,in which IEC proliferation is involved.Polyamines are essential for IEC cell proliferation,and affect the stability and translation of transcripts by regulating the posttranscriptional regulation of RNA-binding protein HuR on target mRNA in IEC cells,and play an important role in maintaining the structural and functional integrity of intestinal mucosa.HuR modified by phosphorylation,methylation or ubiquitination,regulates gene translation by altering subcellular localization and its ability to bind target mRNAs.Depletion of intracellular polyamines by DFMO(difluoromethylornithine,polyamine synthesis inhibitor)significantly increased HuR abundance in the cytoplasm and decreased HuR level in the nucleus,but total HuR content in the whole cell remained unchanged.In addition,depletion of intracellular polyamines also inhibited Chk2(cell cycle checkpoint kinase 2)activity and decreased the affinity of HuR to target gene c-Myc mRNA(nuclear transcription factor).The addition of exogenous polyamines could increase the distribution of HuR in the nucleus,improve the activation state of Chk2 and the phosphorylation level of HuR,enhance the association between HuR and c-Myc mRNA 3’UTR,promote c-Myc translation,which helped to regulate the distribution of IEC-6 cell cycle,promote cell proliferation,and then accelerate the repair of damaged intestinal mucosa.Clinical and experimental studies have confirmed that the Yiqi Jianpi TCM can improve the repair of intestinal mucosa injury.The previous research of our group found that Panax ginseng aqueous extracts(PGE),a kind of Yiqi Jianpi TCM,could prevent and treat indomethacin-induced intestinal mucosal injury in rats,and relieve the symptoms of weight loss,dull hair,and melena in model rats.Total ginsenosides(TG),one of the active components of Panax ginseng,could promote the IEC-6 cell proliferation and increase the expression of cell proliferation regulatory proteins such as c-Myc,CDK2,and RhoA,by affecting the regulation mechanism of polyamines.These studies provide the basis for exploring the mechanism of total ginsenosides in promoting cell proliferation through the HuR posttranscriptional regulation mediated by polyamine.ObjectiveWe will observe the effects of total ginsenosides(TG)on HuR posttranscriptional regulation mediated by polyamines during the intestinal epithelial cell(IEC-6)proliferation.The cell cycle,HuR regulation mechanism and posttranscriptional regulation of target gene c-Myc were detected at the cellular level,and the effect of Panax ginseng on DSS-induced intestinal mucosal injury was studied at the animal level.These will provide a reference for exploring the protective effect of Yiqi Jianpi TCM Panax ginseng on intestinal mucosa.Methods1.Effect of total ginsenosides on cell proliferation-related indicators in different culture conditions(normal culture,containing DFMO,containing HuR inhibitor CMLD-2):MTT assay was conducted to detect cell proliferation;flow cytometry was used to determine cell cycle distribution;qRT-PCR was carried out to detect c-Myc mRNA level;Western Blot was conducted to exam c-Myc protein expression.2.Effect of total ginsenosides on posttranscriptional regulation of HuR in the culture condition containing DFMO:the double-luciferase reporter gene system was used to detect the translation efficiency of c-Myc mRNA;after Actinomycin D blocked gene transcription,qRT-PCR was carried out to measure the c-Myc mRNA level in each group at different time points,which could be evaluated stability of c-Myc mRNA;The level of HuR/c-Myc mRNA complex was determined by RIP(RNA-binding protein immunoprecipitation)method;the abundance of HuR in the nucleus and cytoplasm was detected after extracting nuclear protein/cytoplasmic protein;the nucleoplasmic distribution of HuR was detected by IF(immunofluorescence);IP(immunoprecipitation)was conducted to exam p-HuR expression level;Western Blot was carried out to detect expression of p-Chk2,nonphosphorylated Chk2 and total HuR.3.Effect of Panax ginseng aqueous extracts on intestinal mucosal injury induced by DSS in mice:mice were given 3%DSS for 7 days for modeling.Panax ginseng aqueous extracts(PGE 5g/kg,15g/kg)and positive control drug mesalazine(0.1g/kg)were respectively administered by gavage every day for 7 days;at the same time,the mice in the Control group and the Model group were given pure water.DAI scores(body weight,fecal traits and occult blood)were performed for the mice in each group at d4 and d6.After the experiment,the colon was taken and weighed,the colon length was measured.The colon mass index and unit colon mass index were calculated.Histopathological score was performed under the microscope after H&E staining of colonic mucosa,and the expression of HuR and c-Myc proteins in the colon mucosa were detected by immunohistochemistry.Results1.Effect of total ginsenosides on the cell proliferation:①In normal culture(including serum starvation),the treatment with total ginsenosides(100mg/L)could promote cell proliferation after 4 days of intervention(compared with the Control group,P<0.05),suggesting that total ginsenosides had the effect of promoting cell proliferation.②DFMO(5mmol/L)inhibited cell proliferation(compared with Control group,P<0.01),while the treatment with total ginsenosides(50mg/L or 100mg/L)could reverse the inhibition of cell proliferation caused by DFMO(compared with DFMO group,P<0.01),suggesting that the effect of total ginsenosides on cell proliferation was related to the regulation of polyamines.③ CMLD-2(HuR inhibitor,3μmol/L)inhibited cell proliferation(compared with the Control group,P<0.01),and total ginsenosides had no significant effect on the inhibition of cell proliferation caused by CMLD-2(compared with the CMLD-2 group,P>0.05),suggesting that if HuR was inhibited,the effect of total ginsenosides on cell proliferation was also inhibited.These results suggest that the effect of total ginsenosides on cell proliferation is related to the regulation of polyamines and HuR.2.Effect of total ginsenosides on cell cycle:①In normal culture(including serum starvation),the treatment with total ginsenosides(100mg/L)enhanced the proportion of S+G2/M phase cells and reduced the proportion of G0/G1 phase cells(compared with the Control group,P<0.05),suggesting that total ginsenosides could promote cell mitosis and then increase cell proliferation.②Depletion of intracellular polyamines by DFMO significantly enhanced the ratio of G0/G1 phase cells and reduced ratio of S+G2/M phase cells(compared with the Control group,P<0.05).Total ginsenosides(50mg/L and 100mg/L)could reverse the inhibitory effect of DFMO on cell cycle progression,increase the proportion of cells in S+G2/M phase and decrease the proportion of cells in G0/G1 phase(compared with DFMO group,P<0.05).It was suggested that the effect of total ginsenosides in regulating cell cycle was related to polyamines.③CMLD-2 up-regulated the percentage of cells in G0/G1 phase and down-regulated the percentage of cells in S+G2/M phase(compared with the Control group,P<0.01),and total ginsenosides had no significant effect on the cell cycle arrest induced by CMLD-2,suggesting that if HuR was inhibited,the effect of total ginsenosides in regulating cell cycle was also inhibited.The above results show that the effect of total ginsenosides in promoting cell proliferation is tied up with its regulation of cell cycle and promotion of cell mitosis,as well as thethe regulation of polyamines and HuR.3.Effect of total ginsenosides on the expression c-Myc mRNA and protein:①In normal culture(including serum starvation),the expression levels of c-Myc mRNA and protein in total ginsenosides groups(TG50mg/L,100mg/L)were much higher than that in control group(P<0.05).②There was no significant difference in c-Myc mRNA level after depletion of intracellular polyamines by DFMO(compared with the Control group,P>0.05),but c-Myc protein level was decreased(compared with the Control group,P<0.01);total ginsenosides(50mg/L,100mg/L)could reverse the decreased expression of c-Myc protein induced by DFMO(compared with the DFMO group,P<0.05),but had no significant effect on the c-Myc mRNA expression(compared with the DFMO group,P>0.05),suggesting that the effect of DFMO on c-Myc and the pharmacodynamics of total ginsenosides were mainly related to c-Myc protein expression(posttranscriptional event)rather than mRNA expression(transcriptional level).③CMLD-2 had no significant effect on c-Myc mRNA level(compared with Control group,P>0.05),but the level of c-Myc protein was decreased(compared with Control group,P<0.01),suggesting that HuR mainly regulated c-Myc at the posttranscriptional level but had no significant effect on its transcriptional level;The expression of c-Myc protein in total ginsenosides group was inhibited(compared with Control group,P<0.01;compared with CMLD-2 group,P>0.05),the level of c-Myc mRNA in total ginsenosides group did not change significantly(compared with Control group or CMLD-2 group,P>0.05).It indicated that the effect of total ginsenosides on cMyc was also inhibited when HuR was inhibited.These results indicate that total ginsenosides regulate the expression of c-Myc(proliferation regulatory protein)by affecting the HuR posttranscriptional regulation mediated by polyamine.4.Effect of total ginsenosides on HuR posttranscriptional regulation related indicators in culture containing DFMO:①Effect of total ginsenosides on translation efficiency of c-Myc mRNA:the translation efficiency of c-Myc mRNA was decreased in the DFMO group(compared with the Control group,P<0.05),while the treatment of total ginsenosides(50 mg/L,100 mg/L)reversed the decrease in the translation efficiency of c-Myc mRNA caused by DFMO(compared with the DFMO group,P<0.05).②Effect of total ginsenosides on the stability of c-Myc mRNA:the half-life of c-Myc mRNA in IEC-6 cells was shortened in the DFMO group(the half-life of c-Myc mRNA in Control group was 1h-2h,while that in DFMO group<1h),indicating that DFMO reduced the stability of c-Myc mRNA.The total ginsenosides(25mg/L,50mg/L)could significantly prolong the half-life of c-Myc mRNA(1.5h～2h),suggesting that total ginsenosides enhanced c-Myc mRNA stability and reduced its transcript degradation.③Effect of total ginsenosides on HuR/c-Myc mRNA association:the expression of HuR/c-Myc mRNA complex was decreased in DFMO group(compared with Control group,P<0.01),while total ginsenosides(50mg/L,100mg/L)could significantly increase the expression of HuR/c-Myc mRNA complex(compared with DFMO group,P<0.01),suggesting that total ginsenosides enhanced the affinity of HuR to c-Myc mRNA.④Effect of total ginsenosides on nucleoplasmic distribution of HuR and expression of p-HuR:the whole-cell level of HuR in the DFMO group did not change(compared with the Control group,P>0.05),but the abundance of HuR in the cytoplasm was increased and the distribution in the nucleus was decreased(compared with the Control group,P<0.05);total ginsenosides decreased the content of HuR in the cytoplasm and increased its level in the nucleus(compared with DFMO group,P<0.01);DFMO reduced the expression of p-HuR in cells,while total ginsenosides(25mg/L,50mg/L,100g/L)increased the level of p-HuR,indicating that the inhibitory effect of DFMO was reversed.⑤Effect of total ginsenosides on the expression of p-Chk2 and non-phosphorylated Chk2 proteins:the expression of p-Chk2 in the DFMO group was significantly decreased(compared with the Control group,P<0.01),while the level of non-phosphorylated Chk2 had no significant change(compared with the Control group,P>0.05).Total ginsenosides(50mg/L,100g/L)increased the level of p-Chk2(compared with DFMO group,P<0.01),but had no significant effect on the expression of non-phosphorylated Chk2(compared with DFMO group,P>0.05).The above results indicate that total ginsenosides could improve the translation efficiency of c-Myc mRNA,prolong the half-life of c-Myc mRNA and increase its stability,enhance the expression of HuR/c-Myc mRNA and its affinity,improve the expression levels of p-Chk2 and p-HuR,and regulate the nucleocytoplasmic distribution of HuR;it indicates that the effect of total ginsenosides in promoting cell proliferation is related to the posttranscriptional regulation mechanism of HuR.5.Effect of Panax ginseng aqueous extracts(PGE 15g/kg,5g/kg)on DSS-induced colonic mucosal injury in mice:①PGE reduced the DAI score of model mice(compared with the Model group,P<0.05)and relieved symptoms.②PGE could restore colon length(compared with Model group,P<0.05),reduce colon weight,colon mass index and unit colon mass index(compared with Model group,P<0.01).③ PGE decreased the histopathological score of colonic mucosa in model mice(compared with Model group,P<0.05).④PGE increased the expression of HuR and c-Myc proteins in colonic mucosa of model mice(compared with Model group,P<0.01).The results indicate that Panax ginseng aqueous extracts effectively alleviate colonic mucosal injury induced by DSS,which may be related to the increase of HuR and c-Myc levels in mucosal tissues.ConclusionTotal ginsenosides could promote the IEC-6 proliferation,which is related to the posttranscriptional regulation of HuR mediated by polyamines,including regulating the cell cycle,affecting the posttranscriptional regulation of HuR target gene c-Myc,controlling HuR/c-Myc complex and HuR upstream indicator Chk2;Panax ginseng aqueous extracts could alleviate colonic mucosal injury induced by DSS by increasing the expression of HuR and c-Myc proteins in mucosal tissues;Total ginsenosides are one of the pharmacodynamic components of panax ginseng to maintain intestinal mucosal homeostasis;The results provide a reliable experimental evidence for exploring the protective effect of Yiqi JianPi TCM panax ginseng on intestinal mucosa. |
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