| Aims:The purpose of this study was to evaluate the stool-softening effect of rhubarb-derived free anthraquinones and its five monomers on water deficit-induced constipation in rats,and to explore its possible stool-softening mechanism.Methods:The free anthraquinone extract(RhA)of Rhei Radix Et Rhizoma was obtained by refluxing with ethanol,extracting with dichloromethane,and resolving with methanol.The content of five monomers in RhA was determined by high-performance liquid chromatography.A rat model of water deficit-induced constipation was established to induce constipation.The effects of RhA extract,its monomers and the combination of active monomers were evaluated based on rat constipation model by assessing changes in feacal water content,the number of feacal pellets within 6 h,the weight of feacal pellets within 6 h,colonic expulsion latency,and intestinal terminal propulsion rate were assessed in rats.Using histopathological measurement,ELISA,immunohistochemistry,qPCR,and Western blotting,the stool-softening mechanism of RhA and its monomers was investigated.Results:(1)Aloe-emodin,rhein,emodin,chrysophanol,and physcion constituted94.49%(w/w)of RhA.(2)RhA,aloe-emodin,rhein,and chrysophanol showed stool-softening activity and the combination of aloe-emodin and rhein had the strongest stool-softening effect.They could significantly increase feacal water content(P<0.05),the number of feacal pellets within 6 h(P<0.05),the weight of feacal pellets within 6 h(P<0.05),colonic expulsion latency(P<0.05),and intestinal terminal propulsion rate(P<0.05).(3)Aloe-emodin,emodin,and chrysophanol improved constipation by increasing VIP(P<0.05),SP(P<0.05)and MTL content(P<0.05)in serum of rats.(4)Aloe-emodin,rhein,and chrysophanol upregulated the expression of VIP,CAP1,PKA,CFTR,AQP3,AQP4,and AQP8,decreased the expression of ENa C,NHE3 at the m RNA and protein levels,and reduced the concentration of Na~+-K~+-ATPase in the colon of constipated rats.○5 RhA,aloe-emodin,rhein and chrysophanol can increase the osmotic pressure,sodium ion and chloride ion concentration in colonic fluid of model rats.Conclusions:Aloe-emodin,rhein,and chrysophanol were the stool-softening components of RhA.RhA could upregulate VIP expression,activate the c AMP/PKA pathway,and stimulate the expression of CFTR,restrain the expression of NHE3 and ENa C,thereby leading to hypertonicity in the lumen of colon.Driven by osmotic gradients,water was transferred to the lumen of the colon.In order to adapt to water transport,the expression levels of AQP3,AQP4 and AQP8 were upregulated passively.In addition,RhA improved gastrointestinal motility and accelerated stool excretion by increasing the serum levels of VIP,SP,and MTL. |
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