The Mechanism Of Buzhongyiqi Decoction For The Treatment Of Spleen Deficiency Diarrhea By Intestinal SGLT1/NHE3 Regulation

ObjectiveToinvestigate the effect and molecular mechanism of BuzhongYiqi Decoction and Atractylenolide I in spleen deficiency diarrhea.Buzhongyiqi decoction come from the book ofdifferentiation on endogenous and exogenous diseases,the decoction is usually used to improve spleen qi deficiency and subsidence.Atractylodes is one of the main drugs of Buzhong Yiqi Decoction Atractylenolide I is effective components of Atractylodes.Study have found that BuzhongYiqi Decoction andAtractylenolide I can promote theability of digestion and absorption of patients and/or animals with spleen deficiency diarrhea.In this study,we established spleen deficient diarrhea Rats and Caco-2 cells models to investigate glucose absorption disorder of Small intestine and the variety of SGLT1/NHE3 pathway,and explored the regulatory mechanism of BuzhongYiqi Decoction and Atractylenolide in improving spleen deficient diarrhea.MethodsExperimental study used "rhubarb + reserpine + control diet" method to set up spleen deficient diarrhea model.the rats was divide into low,medium and high dose group of BuzhongYiqi Decoction and spleen deficient diarrhea model group.General status,diarrhea index,D-xylose excretion rate,glucose uptake content and mucosa histopathological,intestinal Na+-K"ATP enzyme activity was measured.RT-PCR methed was used to detect sodium-dependent glucose transporter(SGLT1),glucose transporter 2(GLUT2),sodium hydrogen exchanger 3(NHE3)mRNA expression levels.We used Westernblotmethed to detect sodium-dependent glucose transporter(SGLTl),glucose transporter 2(GLUT2),sodium hydrogen exchanger 3(NHE3)、P38MAPK、p-P38MAPK、MAPKAPK2、p-MAPKAPK2、AKT2、p-AKT2、EZRIN、p-EZRIN protein expression levels.The influences of Atractylenolide I on SGLT1/NHE3 and MLCK/MLC pathway:Using Phlorizin,SB203580,NSC668394 Block the SGLT1、P38MAPK andEzrin protein,and then adding Atractylenolide I to observe its effects on the expression of NHE3,MLCK protein and MLC phosphorylation by using the Caco-2 cell model.Results1.Before setting up the model,the body weights and urinary D-xylose excretion has no significant difference between the control group and the model group(P>0.05),After the model was finished,the body weight and urinary D-xylose excretionof the model group decreased significantly compared with the normal group(P<0.01).After treating with BuzhongYiqi Decoction,the body weight and urinary D-xylose excretionof treatment groups wereincreased,but the difference of body weight was not statistically significant(P>0.05),while the increase of urinary D-xylose excretion was most significant in high dose group(P<0.01).2.Thediarrhea index were increased,and the glucose uptake were decreased in model group(P<0.05);after giving BuzhongYiqiDecoction,the diarrhea symptoms were gradually alleviated,Both diarrhea indexand the glucose uptake were imporved in a dose-dependent manner,which was most obvious in the high-dose group(P<0.05 or P<0.01).3.Small intestinal mucosahistopathological and Na+-K+ ATPase activity change:Intestinal villus partiallydamaged,epithelial cells absent with inflammatory cells infiltration in the model group,the height and width of the small intestine villus significantly decreased(P<0.01).The Na+-K+ ATPase activity in intestinal mucosa also decreased significantly in the model group(P<0.01),while BuzhongYiqi Decoction could promote small intestinal mucosal repair,increase the height and width of villus(P<0.01)and improve Na+-K+ ATPase activity in a dose-dependent manner(P<0.01).4.The mRNA and protein expression of SGLT1,GLUT2,NHE3 significantly decreased in the model group(P<0.01 or P<0.05),while no significant differences were seen for expression of P38MAPK、MAPKAPK2、Akt2、Ezrin in the P38MAPK/Ezrin pathway,the protein expression level of P-P38MAPK,P-MAPKAPK2,P-Akt2 and P-Ezrin decreased compared withthe control group,after treating with BuzhongYiqi Decoction,the mRNAlevel ofSGLT1,GLUT2,NHE3 and the protein expressions level of SGLT1,GLUT2,NHE3,P-P38MAPK,P-MAPKAPK2,P-Akt2 and P-Ezrin significantly higher than that ofthe model,which was most obvious in the high-dose group(P<0.01orP<0.05).5.The protein and mRNA expressions of SGLT1、NHE3 decreased,and the phosphorylation of P38MAPK/Ezrin reduced after using Phlorizin(P<0.01 or P<0.05),Atractylenolide I could increase the mRNA or protein expressions level of SGLT1,NHE3,P-P38MAPK,P-MAPKAPK2,P-Akt2 and P-Ezrin(P<0.01or<P<0.05).NHE3mRNA and protein expressions decreased after using SB203580 or NSC668394 for blocking P38MAPK or Ezrin,while increased after giving Atractylenolide I,indicating that it probably not promote the expression of NHE3 by P38MAPK and Ezrin.6.The mRNA and protein expressions of MLCK decreased and the phosphory lation of P-MLC reduced after using Phlorizin(P<0.01 or P<0.05),Atractylen olide I could increase the protein expressions level of MLCK and P-MLC(P<0.05).MLCK and P-MLC protein expressions alsodecreased after using SB203580 for blocking P38MAPK,while increased after giving Atractylenolide I,indicating that it probably not promote the expression of MLCK and P-MLC by P38MAPK.Conclusion1.The spleen deficiency diarrhea Rats had lower body weight,urinary D-xylose excretion rate,the glucose uptake,and higher diarrhea index,they also appeareddamage of Intestinal villus,deficiency of epithelial cells and decrease of Na+-K+ ATP enzyme activity.BuzhongYiqi Decoction could improve the general symptoms of spleen deficiency diarrhea rats,reduce diarrhea index,increase body weight,D-xylose excretion rate and the glucose uptake,promote small intestinal mucosal repair and the activity of Na+-K+ATP enzyme of intestinal mucosa.2.The mRNA and protein expression level of SGLT1,GLUT2,NHE3 were decreased in spleen deficiency diarrhea rats,and the phosphorylation of P38 MAPK/Ezrin pathway was Also decreased,while BuzhongYiqi Decoction could increase The mRNA and protein expression level of SGLT1,GLUT2 and thedegree of phosphorylation of P38MAPK/Ezrin pathway,and then promote the expression of NHE3,which might be one of the mechanism ofBuzhongYiqi Decoction for the treatment of spleen deficiency diarrhea.3.The mRNA and protein expressions of SGLT1,NHE3 and the phosphorylation of the P38MAPK/Ezrin pathway decreased after using Phlorizin in Caco-2 cells,The mRNA and protein expressions of NHE3 decreased after blocking P38MAPK or Ezrin.Atractylenolide I could increase the expressions level of SGLT1 and the phosphorylation of the P38MAPK/Ezrin pathway,and then promote the expression of NHE3.4.The mRNA and protein expressions of MLCK decreased,and the phosphorylation of MLC reduced after block SGLT1 or P38MAPK in Caco-2 cells,Atractylenolide I could increase the expressions of MLCK and the phosphorylation of MLC by promoting the expressions of SGLT1.