Studies On Effects Of Intestinal Calcium Transport And Mechanisms Of Anti-diarrhea Of Magnolol And Honokiol

As a second messenger in cellular signal transduction, calcium plays an important role on the regulation of transmembrane delivery of biological signals, glandular secretion; maintain acid-base balance in the body and other physiological activities. As the two main active ingredients in Chinese medicine magnolia officinalis, magnolol and honokiol have a significant effect of anti-diarrhea, but the mechanisms are unknown. Researches showed that there is closely relationship between the intestinal calcium transport and the anti-diarrhea effect, magnolol and honokiol can be calcium channel blockers to inhibit the abnormal intestinal ion transport, yet there is no exact report about their effects of intestinal calcium transport and the mechanism of anti-diarrhea.In this paper, we use magnolol and honokiol as the test materials, through electric physiological test, and cells culture in vitro and gene expression test, with an aim to study effects of different doses of magnolol and honokiol on intestinal short-circuit current, the concentration and gene expression of calcium of IEC-6, and tries to discuss the mechanisms of anti-diarrhea from the point of view of intestinal calcium transport, which provides vigorous scientific evidence on magnolol and honokiol to be new additive possessing the function of anti-diarrhea of livestock and the efficient utilization of Magnolia officinalis. The main research methods and results are as follows:(1) Effects of magnolol and honokiol on short-circuit current of rat ileum and their anti-diarrhea mechanismsTo study the effects of magnolol and honokiol on the short-circuit current of rat isolated ileum, we use ussing chamber system to test and verapamil was the positive control drug. Results showed that the inhibition effects of magnolol and honokiol in rat ileum of short circuit current is significant difference compared with that of verapamil(P<0.01); which can also significantly inhibit the increasing of the short circuit current of acetylcholine activated(P<0.01). we can conclude that there are more kinds of calcium channels inhibited by magnolol and honokiol than verapamil in rat ileum, or both of them not only can inhibit the transport of calcium but also inhibit the transport of other ions, they both may be calcium channel blockers to played a role of anti-diarrhea.(2) Effects of magnolol and honokiol on the concentration of free calcium of IEC-6and their anti-diarrhea mechanismsDifferent concentrations of magnolol and honokiol were used to treat rat intestinal epithelial cells cultured in vitro, we studied effects of magnolol and honokiol on cell viability and choose the suitable concentrations of them(magnolol,5μmol/L、10μmol/L、20μmol/L and honokiol,1μmol/L>5μmol/L、10μmol/L)and test the changes of intracellular free calcium concentration after treating with them using fluorescent labeling method, and also test effects of the increasing of calcium concentration induced by potassium chloride and acetylcholine, to discuss their anti-diarrhea mechanisms. Results showed that magnolol and honokiol can reduce calcium concentration of IEC-6on the resting state, and the inhibition rate increased with the concentrations increase; after pretreating with magnolol and honokiol for5minutes, they can also block the calcium increase due to both50mmol/L KCL and10μmol/L acetylcholine. From this we can know that magnolol and honokiol can influence the calcium transport of IEC-6in various degrees, and control the opening and closing of the voltage-gated calcium channel and receptor operated calcium channel to regulate the calcium flow outside and calcium release to influence the intracellular free calcium concentrations and achieve the effect of anti-diarrhea.(3) Effects of magnolol and honokiol on the gene expression of calcium of IEC-6and their anti-diarrhea mechanismsWe use real-time fluorescent quantitative PCR techniques to study the effects of different concentrations of magnolol and honokiol on the gene expression of TRPV6, PMCAlb, NCX-1, CACNA1C, CACNA1D, KCNMA1, KCNN1, KCNN2, KCNN3and KCNN4of IEC-6. Results showed that magnolol and honokiol can increase the expression of TRPV6, have some effects on the expression of PMCAlb and NCX-1; there is an significant difference of CACNA1C and CACNA1D of high doses of them, also have some effects of KCNMA1, KCNN1and KCNN2while have a double action of KCNN3and KCNN4. We can know that magnolol and honokiol can combine with L-type voltage-gated calcium channel, inhibit its opening and closing and reduce the flow of extracellular Ca2+; at the same time they activate Ca2+-ATPase in plasmalemma to activate the exchange of sodium and calcium, influence the opening and closing of calcium activated small conductance potassium channel and gene expression and receptor operated calcium channel to have an effect of the calcium transport to anti-diarrhea.