| Objective:To study the effects and mechanism of Faeces Bombycis on the expression of aquaporin in rats with syndrome of damp retention in middle-jiao,to clarify the mechanism of “dampness-resolving”,to expand the clinical application of Faeces Bombycis as a “dampness-resolving” drug,and to reveal its scientific connotation,promotes the inheritance and development of traditional Chinese medicine in feces.Methods: 1.Study on the regulation of aquaporin expression by Faeces BombycisThe rats were used to establish the syndrome of “damp retention in middle-jiao” model by the method of “outer damp invades+ righteous gas is exhausted+ eating overfull fat and sweetmeat”,the expression of protein and m RNA of AQP1 in renal and lung tissue,AQP3 in colon and skin tissue,AQP5 in submandibular gland tissue were detected by Western blot and qPCR.The effects of Faeces Bombycis on the expression of aquaporin in rats with the syndrome of damp retention in middle-jiao were investigated.2.Mechanism of Faeces Bombycis regulating aquaporin expressionUsing Caco-2 cells as a model,applying signal pathway agonists and inhibitors,and using RNA interference technology to investigate the effect of cAMP-PKA-CREB signaling pathway on the expression of aquaporin,and to explore the cAMP-PKA-CREB signaling pathway in Faeces Bombycis regulating aquaporin expression.3.Basic research on pharmacodynamic substance of Faeces Bombycis regulating aquaporinThe Caco-2 cell model was used for uptake experiments,and ultra-high performance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry(UPLC-Q-TOF-MS)was used to analyze the changes in intracellular substances before and after ingestion.Peak View software was used to determine the cellular uptake.The composition of the components clarifies the material basis of Faeces Bombycis regulating aquaporins.Results:1.The expression of protein and m RNA of AQP1 in renal and lung tissue,AQP3 in colon and skin tissue,AQP5 in submandibular gland tissue were detected by Western blot and qPCR.Compared with the normal group,the expression of AQP1 protein and m RNA in renal tissue in the model group was significantly increased(P<0.05),the expression of protein and m RNA of AQP1 in lung tissue,AQP3 in colon and skin tissue,AQP5 in submandibular gland tissue in the model group were significantly decreased(P<0.05);compared with the model group,the expression of AQP1 protein and m RNA in renal tissue in the high dose group was significantly reduced(P<0.05),and the protein and m RNA expression of AQP1 in lung tissue,AQP3 in colon and skin tissue,AQP5 in submandibular gland tissue in the model group were significantly increased(P<0.05).2.The effects of signal pathway agonists and inhibitors on the expression of AQP m RNA in Caco-2 cells were detected by qPCR.The results showed that compared with the blank control group,the m RNA expression of AQP3 in the Faeces Bombycis group was significantly increased(P<0.05);after the AC inhibitor SQ22536,cAMP agonist 8-Br-cAMP,and PKA inhibitor H-89 were given,compared with the blank control group,the expression of AQP3 m RNA in the 8-Br-cAMP group was significantly increased(P<0.05),while the expression of AQP3 m RNA in the SQ22536 group and the H-89 group were decreased(P>0.05).Compared with the SQ22536 group,the expression of AQP3 m RNA in the SQ22536+ Faeces Bombycis group was significantly increased(P<0.05);compared with the H-89 group,the expression of AQP3 m RNA in the H-89+ Faeces Bombycis group was significantly increased(P<0.05);and compared with the 8-Br-cAMP group,the expression of AQP3 m RNA in the 8-Br-cAMP+ Faeces Bombycis group was significantly increased(P<0.05).The qPCR method was used to detect the effect of cell transfection si RNA on the m RNA expression of AQP3.Compared with the blank control group,the m RNA expression of AQP3 in the Faeces Bombycis group was significantly increased(P<0.05),and AQP3 m RNA expression in the si RNA-CREB 100 n M group was significantly reduced(P<0.05).There was no significant difference in AQP3 m RNA expression in the si RNA-NC group(P>0.05).Compared with the si RNA-CREB 50 n M group,the expression of AQP3 m RNA in the si RNA-CREB 50 n M+ Faeces Bombycis group was increased(P>0.05);compared with the si RNA-CREB 100 n M group,the expression of AQP3 m RNA in the si RNA-CREB 100 n M+ Faeces Bombycis group was increased(P>0.05).3.A total of 4 compounds were detected and analyzed from the cell uptake experiments,including 2 alkaloid compounds fagomine,3-epi-fagomine,and 2 flavonoid compounds astragalin and quercetin-3-rhamnoside.Conclusions:Faeces Bombycis regulates the expression of aquaporin in the tissues of renal,lung,colon,skin and submandibular glands,and exerts a “dampness-resolving” effect,and the regulation of aquaporin may be related to the fagomine,3-epi-fagomine,astragalin and quercetin-3-rhamnoside are involved in regulating the cAMP-PKA-CREB signaling pathway. |
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