Effects Of Astragalus Membranaceus On Glucose Absorption In Intestinal Epithelial Cells And Its Regulatory Mechanism

Backgrounds:Transportation isone of the main physiological function of spleen, and dys-splenism can induce gastrointestinal digestion and absorption dysfunction, what’s more systemic manifestations, such as abdominal distension diarrhea, fatigue, obstacles absorption of nutrients is outstanding performance. Astragalus Membranaceus(HQ) is a common used drug for the treatment of spleen-deficiency in Chinese medicine. Modern pharmacological studies have shown that Astragalus Membranaceus play an important role in improving gastrointestinal mucosal morphology, intestinal digestion and absorption of spleen-deficiency animal model.Glucose is one of the three major nutrients required for human life activities, it plays an important role in body composition and energy metabolism. Glucose is a polar molecule, it can not freely diffusing through the cell membrane lipid bilayer structure, its transportion needs mediated transporter. And glucose absorption in the intestinal cells mainly through two ways:The first is the sodium-dependent glucose transporter SGLTl-mediated sodium-dependent active transportion. Another approach is facilitated diffusion, the transporter is glucose transporter type 2--GLUT2. Previous studies showed that glucose absorption decrease and low expression of GLUT2 inspleen-deficiency animal model and Panaxand sijunzi decoction can improve the phenomenon of spleen-deficiency animal model.Astragalus Membranaceusreplenish qi to invigorate the spleen, the one of presendent of Qi Tonifying drug and usually used to treat weakness of the spleen and the stomach, transport disorders, poor appetite, loose stools and so on. Modern studies have shown that Astragalus Membranaceus contains saponihs, flavonoids,polysaccharides, trace elements and other compounds, these components have different pharmacological activities, it can be divided into the following categories:gastrointestinal mucosa repair, immune function, anti-aging effects,antibacterial and antiviral and anti-tumor, etc. Astragalus Membranaceus can significantly improve gastric mucosal injury in patients of spleen-deficient, promote the intestinal absorption of nutrients, but the mechanism and material basis are still not clear.Our study will comprehensively explore the significance of Astragalus Membranaceus in regulating glucose absorption and mechanism in spleen-deficiency animal model and Caco-2 cell. Providing scientific clues to explore the nature of spleen-deficiency syndromen in terms of the absorption of nutrients, and to provide a clear target for replenishing qi to invigorate the spleen recipe.Objective:Through observing the changes of glucose absorption and expression of SGLT1 and GLUT2 induced by Astragalus Membranaceus to discuss the mechanism.Methods:1.Copy spleen deficient rat model by subcutaneous injection of reserpine. And then using Astragalus membranaceus solution lavage treatment. observe the improvement of general symptoms and glucose absorption, the pathological changes of intestines, western-blot and PCR detect the expression SGLT1 and GLUT2.2. Establish Caco-2 cells glucose absorption detection method, follows previous Caco-2 cells planting density, Screening best concentration and incubation time.3. Extraction and separation of effective parts of astragalus root by solvent extraction. The effect of the extraction of Astragalusextracts on Caco-2 cell model was obtained. To screen effective parts of glucose uptake in Caco-2 cells.4. The optimal dosage of the effective part of Astragalus is screened out to promote the glucose uptake of Caco-2 cells. In the glucose uptake model of Caco-2 cells, the regulation of glucose uptake and related transporters were observed in the case of adding or not adding the related pathway inhibitors.5.To observe Gaco-2 cells’gluose uptake and glucose transporter expression under the intervention of PZ (SGLTl inhibitor), PT(GLUT2 inhibitor), U73122(Phospholipase inhibitor), PMA(PKC agonist). To rearch the effect of Astragalus membranaceus n-butyl alcohol extracted part in Caco-2 cells’gluose uptake and its regulatory mechanism.Results:1. Successfully reproduce the rat model of spleen-deficient. Model group rats appeared listlessness, loose stools, less activity, etc. Compared with control the weight and D-xylose excretory rate were decrease. In vivo perfusion indicated that glucose uptake is reduced. Western-blot showed down-regulation of SGLTl and GLUT2. After treatment with water extraction of astragalus, Compared with model control, the general state, weight, D-xylose excretory rate and glucose absorption were improved. What’s more the water extraction of astragalus induced the up-regulation of SGLTl and GLUT2 both protein level and gene level.2. Established Caeo-2 cells glucose absorption detection method:The 2-NBDG was used to detected glucose absorption. The best concettration and time of incubation are 100 μmol/L and 40 min respectively.Astragalus membranaceus n-butyl alcohol extracted parts for promoting glueose absorption effect is best and effective concentration range is 50mg/L-175mg/L.3. Caco-2 cells were treated with increased doses of Astragalus membramceus n-butyl alcohol extracted parts(50mg/L、100 mg/L and 150mg/L), then 2-NBDG detected glucose absorption, the result showed that the Astragalus mmbranaceus n-butyl alcohol extracted parts can promote glucose absorption in a dose-dependent manner.4. The Astragalus n-butyl alcohol extracted parts can up-regulation SGLT1 and GLUT2 expression on protein level and gene level in a dose-dependent manner.5. Inhibitor PZ can reduce absorption of glucose obviously, and Astragalus n-butyl alcohol extracted parts (150mg/L) can reverse the effect of PZ. PT had no impact on glucose absorption without Astragalus n-butyl alcohol extracted parts. But PT can weaken the effect of Astragalus n-butyl alcohol extracted parts (150 mg/L) on Caco-2 cell glucose absorption.6. Under the intervention of PZ, the expression of SGLT1 was down-regulated; same situation, PT down-regulated GLUT2 protein’ expression. Astragalus Membranaceus n-butyl alcohol extracted parts (150 mg/L) can improve the expression of SGLT1 and GLUT2 protein expression.7. Under the intervention of U73122, the effect of Astragalus Membranaceus n-butyl alcohol extracted parts on Caco-2 cell glucose absorption and GLUT2 expression is weakened. PMA and Astragalus Membranaceus n-butyl alcohol extracted parts have the same effect on Caco-2 cell glucose absorption and GLUT2 expression.Conclusion:1.SGLT1 and GLUT2 down-regulation may be involved in Glucose malabsorption of spleen-deficiency rats, and Astragalus Membranaceus can improve glucose malabsorption through regulating the two protein expression.2. Astragalus can improve glucose absorption of Caco-2 and theAstragalus Membranaceusn-butyl alcohol extracted part is the most active ingredients.3. GLUT2 may be the main protein in regulating glucose absorption and the target of Astragalus Membranaceus n-butyl alcohol extracted parts.The Astragalus Membranaceus n-butyl alcohol extaracted part is probably mainly through the PKC-Ca2+-GLUT2 pathway, which promotes the glucose uptake of Caco-2 cells.