Based On SGLT1 Pathways To Study The Effects Of Sijunzi Decoction On Caco-2 Glucose Uptake

ObjectiveThe domestic studies for years have suggested that patients with syndrome of spleen-qi deficiency exists intestinal mucosal epithelial cell damage and the reduced glucose absorption. Replenishing qi to invigorate the spleen method is the fundamental rule of traditional Chinese medicine treatment of spleen deficiency syndrome, it has obvious effect on gastrointestinal mucosal protection and glucose absorption barriers improved in spleen deficiency syndrome. This paper from in vitro cultured cells, observe the polyamine effect of Caco-2 cells to absorb glucose and sodium-dependent glucose transporter 1 (SGLT1) pathway, and to explore relations between the mechanism of sijunzi decoction functions of spleen invigorating and transportation and polyamine of SGLT1 pathway mediates effect on glucose absorption, to clarify the spleen transport and transform nutrients and replenishing qi to invigorate the spleen method and its action mechanism of the formulas provide a clear target.Methods1 the establishment on the Caco-2 cells modelCaco-2 cell proliferation was detected by MTT method; Caco-2 was cultured for 21 days to observe morphology and microvillus; cell resistance meter detected transmembrane resistance (TEER).2 the effect on Caco-2 cell membraneRats was divided into 2 groups, black control group and sijunzi decoction group. Rats were gavaged sijunzi decoction with 17 g/kg, blank control group was given the equivalent normal saline,2 times/d,3 d in a row, after time for 1 h at the end of the gavaged, draw blood from abdominal aorta for si junzi decoction medicated serum blood preparation. Caco-2 cell proliferation and inhibition was detected by MTT method; microvillus were examined with electron microscope.3 the effect on Caco-2 cell glucose uptake and consumptionGlucose determination kit and 2-NBDG is used to detected Caco-2 cells glucose uptake and consumption.4 the effect on SGLT1 signal pathways of Caco-2 cellRT-PCR and Western blot examined SGLT1 and GLUT2 mRNA and protein expression changes; Flow Cytometer (FCM) detected cell [Ca2+] cyt level; Western blot detected Caco-2 P-MLC protein.5 the effects on polyamine content of Caco-2 cellsthe polyamines in Caco-2 cell was determined by pre-column derivation high performance liquid chromatograph.Results1 the establishment on the Caco-2 cells modelDetermined by MTT method in the growth curve showed that cells was in logarithmic growth phase in a 2 d~6 d, cell morphology observation found that cells has basically formed a tight cell monolayer in about 15~16 days; the cultivation of 21 d Caco-2 cells were examined with electron microscope that cells had the microvillus; cell resistance meter is used to test different time Caco-2 cell TEER, found that resistance has been> 500 Ω · cm2 and cell monolayer has formed in about 15 ~ 16 d.2 the effect on Caco-2 cell membraneMTT result show that DFMO obviously inhibited Caco-2 cell proliferation (P<0.05), SPD and SJZTeach dose group could improve DFMO inhibitory effect(P<0.05, P<0.01), and promote Caco-2 cell proliferation; electron microscope observation result show that the microvillus of DFMO group was obviously shorter than the control group with irregular and broken(P<0.05, P<0.01). The SPD and SJZT group can improve DFMO inhibition for the growth of microvillus (P<0.05, P<0.01).3. the effect on Caco-2 cell glucose uptake and consumptionGlucose detection kits results show that SPD and SJZT could promote the normal Caco-2 cells of glucose consumption, improve the inhibition of Caco-2 cells glucose consumption by DFMO and phloridzin and promote the absorption of glucose; observed by inversion fluorescence microscope showed that the Caco-2 cells can effectively absorb 2-NBDG, SPD and SJZT promoted the normal Caco-2 cells to absorb glucose(P<0.05, P<0.01), and as SJZT concentration increases, the effect of the glucose uptake was more and more strong; besides SPD and SJZT improved the inhibition of Caco-2 cells glucose uptake by DFMO and phloridzin (P<0.05, P<0.01).4 the effect on SGLT1 signal pathways of Caco-2 cellRT-PCR and Western blot results show that SGLTland GLUT2 mRNA and protein leves of the DFMO or phloridzin were lower than the control group(P<0.05, P<0.01), SPD and SJZT can increased normal cells SGLT1 and GLUT2 mRNA and protein expression level(P<0.05, P<0.01), and also can reverse inhibition on SGLT1, GLUT2 mRNA and protein expression of the phloridzin and DFMO, significantly improve the level of the expression of glucose transporter protein, and 10% SJZT effect is better; FCM results show that the [Ca2+] cyt level of phloridzin and DFMO groups significantly lower than the control group, SPD and SJZT can improve the level of intracellular [Ca3+] cyt (P<0.05, P<0.01), and also can reverse inhibition of the phloridzin and DFMO(P<0.05, P<0.01), increase the concentration of intracellular [Ca2+] cyt; the results of Western blotting showed that, P-MLC protein levels of phloridzin and DFMO group were significantly lower than the control group(P<0.05, P<0.01); SPD and SJZT reversed the inhibition of the P-MLC protein expression of phloridzin and DFMO, improve the level of P-MLC protein expression(P<0.05, P<0.01).5 the effects on polyamine content of Caco-2 cellsHPLC results show that the Caco-2 cell cell polyamine (putrescine, spermidine and spermine) content of DFMO group were much lower than the control group(P<0.05, P<0.01), the SPD and SJZT can not only improve the normal Caco-2 polyamine content in cells, also can reverse reduction of polyamine content in cells by DFMO caused(P<0.05, P<0.01), improve the polyamine content in cells.Conclusion1 the establishment on the Caco-2 cells modelCaco-2 cell model of bing cultured in our laboratory can be used as a study of small intestine absorption in vitro model, and can be carried out the experiment in 15~16 d. 2 the effect on Caco-2cell membraneStructure influence function, improving or promoting intestinal mucosal epithelial cell proliferation and the change of the structure is associated with cell glucose uptake, may be the mechanism of sijunzi decoction in the treatment of spleen-qi deficiency by improving intestinal absorption function, repair the damaged intestinal tissue cells, and may influence the expression of glucose related transport carrier, it remains to be further research.3 the effect on Caco-2cell glucose uptake and consumptionPolyamine and sijunzi decoction can enhance Caco-2 fluorescence that inhibited by DFMO and phloridzin, promote the absorption of glucose. It was connected with glucose transporter of Caco-2 cell, and polyamine and sijunzi decoction may through influence glucose transporters to mediate glucose absorption.4 the effect on SGLT1 signal pathways of Caco-2 cellThe effect of polyamine and sijunzi decoction to promote Caco-2 cell proliferation and glucose absorption was related to promote SGLT1 and GLUT2 protein expression, which is Closely relate to the polyamine synthesis; polyamine and sijunzi decoction can through increasing polyamine content in cells, and improve [Ca2+] cyt and P-MLC protein level to promote glucose absorption that mediates by SGLT1 pathway, in order to promote Caco-2 cell proliferation and improve intestinal mucosal repair function.5 the effects on polyamine content of Caco-2 cellsPolyamine and sijunzi decoction can promote Caco-2 cell proliferation and glucose absorption, improve the structure of the cell microvillus; polyamine regulate cells glucose absorption mediated by SGLT1 pathways and sijunzi decoction can improve the polyamine content, thus prove that the mechanism of sijunzi decoction improving gastrointestinal mucosal damage and glucose absorption barriers is related to the effect of polyamine regulate glucose absorption of SGLT1 mediating, also is the important mechanism for the treatment of glucose absorption barriers of spleen-qi deficiency syndrome. Moreover it provides a very meaningful experimental data for the mechanism of intestinal mucosal repaired by replenishing qi to invigorate the spleen method.