| BackgroundThe gastrointestinal mucosa injuries is one of the pathological basis of gastrointestinal disease, Gastrointestinal mucosa has a selfrepairing capability, the healing process included cell migration, cell proliferation, cell apoptosis and mucosal remodeling. The process of early intestinal epithelial restitution refers to resealing of superficial wounds after injury and occurs as a consequence of epithelial cell migration into the defect, and the process is stimulated by a wide range of divergent factors, including polyamines, trefoilproteins, mucins, growth factors and prostaglandins, etc. the studies on intestinal epithelial cells (IEO6) shows that polyamines signaling pathways is the key controlled factors in the intestinal epithelial cell migration. In the process of intestinal epithelial cell migration after wounding, the upstream index, including polyamines, K+ channel protein and cell membrane potential, raise cytosolic free Ca2+([Ca2+]cyt) through enhancing the driving force for Ca2+ influx, resulting in IEC-6 cell migration during restitution. The regulation index of intracellular calcium stores such as Phospholipase C-γ1 (PLC-γ1) and Inositol triphosphate(IP3), regulate [Ca2+]cyt, in addition, calcium channel protein such as transient receptor potential channel 1 (TRPC1) also regulate [Ca2+]cyt, resulting in activating the downstream targets of calcium, including Rho GTPase, cytoskeletal protein myosin II,etc. which causes intestinal epithelial cell migration. An increasing body of evidence indicates that calcium signaling pathway plays an important role in polyamines mediating intestinal epithelial cell migration.Pathological changes of gastrointestinal mucosal injuries could be observed in spleen deficiency patients, nourishing qi to invigorate spleen is the main way to treat spleen-deficiency syndrome. Sijunzi Decoction is a representative formula of Yiqi Jianpi herbs, which has efficacy on gastrointestinal mucosal injuries restitution. The research results in our group showed that the extracts (such as polysaccharides extracts, flavonoids extracts, etc.) of Yiqi Jianpi herbs, including Astragalus, Atractylodes macrocephala, Codonopsis pilosula, Glycyrrhiza, promoted IEC-6 cell migration and the underlying mechanism was related to the effect on polyamines signaling pathway, Polysaccharides was one of main efficacy component of Yiqi Jianpi herbs on cell migration.ObjectiveTo observe the effect of Sijunzi Decoction polysaccharides on polyamines mediating calcium signaling pathways during IEC-6 cell migration. Investigate the underlying mechanism of Yiqi Jianpi herbs representative formula Sijunzi Decoction on promoting gastrointestinal mucosal injuries restitution, Providing scientific basis for therapeutic mechanism of Sijunzi Decoction on Treatment of gastrointestinal injuries related diseases.Methods(1) Extraction, separation, purification of Sijunzi Decoction polysaccharides:Sijunzi Decoction total polysaccharides was obtained by water extraction and ethanol precipitation method. Eliminating protein by adding Sevag reagent into total polysaccharides to get crude polysaccharides. Crude polysaccharides were purified with DEAE-52 cellulose column chromatography, and then obtained purified polysaccharides. Phenol-sulfuric acid method was used for detection of the content of Sijunzi Decoction total polysaccharides, crude polysaccharides and purified polysaccharides. The purity of Sijunzi Decoction purified polysaccharides were analyzed by HPLC.(2) Selection of tested drug and groups of experiments:Effect of Sijunzi Decoction total polysaccharides, crude polysaccharide and purified polysaccharides on cell migration were observed in pharmacological model of IEC-6 cell migration, and the best effectiveness polysaccharides was selected as the tested drug of our research. IEC-6 cell experiment was divided into five groups, including normal control group, spermidine positive control group, Sijunzi Decoction purified polysaccharides 40,80,160 mg·L-1 groups (in test loaded with 4-AP, tested drug were divided into polysaccharides 20,40,80 mg ·L-1 groups). Stressed test with DFMO(polyamines synthesis inhibitor) or 4-AP(potassium channel inhibitor) was divided into six groups, including normal control group, DFMO or 4-AP model group, spermidine positive control group and tested drug groups with DFMO or 4-AP.(3) Cell migration experiments:IEC-6 cell migration model was established by scratch wound. Cell migration after wounding was observed by phase contrast microscope.(4) The content of cell polyamines, including spermidine and spermine, were measured by HPLC.(5) The mRNA expression levels of Kvl.1, PLC-γ1, TRPC1 were determined by RT-qPCR, respectively.(6) The protein expression levels of Kvl.1, PLC-γ1, TRPC1, RhoA, Racl, Cdc42 were detected by western blot, respectively.(7) The cytosolic free Ca2+ concentration([Ca2+]cyt) and cell membrane potential were detected by flow cytometry, respectively.(8) The Inositol triphosphate(IP3) was detected by enzyme-linked immunosorbent assay(ELISA).(9) The protein expression levels of GTP-RhoA, GTP-Rac1,GTP-Cdc42 were measured by pulldown assay, respectively.(10) The protein expression level of myosin Ⅱ was measured by immunofluorescence assay.Results(1) Extraction, separation, purification and tracking activity of Sijunzi Decoction polysaccharides: ①The extraction rate of Sijunzi Decoction total polysaccharides, crude polysaccharide and purified polysaccharides were 5.42%, 4.19%,2.44%, respectively. ② The content of Sijunzi Decoction total polysaccharides, crude polysaccharides and purified polysaccharides were 67.9%,72.4%,81.6%, respectively. ③ The result of polysaccharides tracking activity indicated that three Sijunzi Decoction polysaccharides all promoted cell migration, the effect of Sijunzi Decoction purified polysaccharides on cell migration more significantly, thus purified polysaccharides was selected as the tested drug in present study. The experimental dose was ranged from 20 mg·L-1 to 160 mg·L-1, the effective dose was ranged from 40 mg·L-1 to 160 mg·L-1.(2) Effect of Sijunzi Decoction polysaccharides on IEC-6 cell migration: Sijunzi Decoction polysaccharides increased cell migration and reversed the inhibitory effect on cell migration inhibited by DFMO(polyamines synthesis inhibitor). These foundings indicated that Sijunzi Decoction polysaccharides promoted cell migration, which involved regulation on polyamines.(3) Effect of Sijunzi Decoction polysaccharides on upstream index of polyamines mediated calcium signaling pathway during cell migration:①Sijunzi Decoction polysaccharides enhanced the content of intracellular polyamines, including spermidine and spermine, and reversed low intracellular spermidine and spermine levels induced by DFMO. ② Sijunzi Decoction polysaccharides increased Kvl.1 mRNA and protein expression levels, and recovered low levels of Kvl.1 mRNA and protein caused by DFMO to normal levels. ③ Sijunzi Decoction polysaccharides reversed cell migration, low levels of Kvl.1 mRNA and protein expression inhibited by 4-AP.④Sijunzi Decoction polysaccharides increased cell membrane potential resulting in membrane potential hyperpolarization, and reversed cell membrane potential depolarization induced by DFMO. These findings indicated that Sijunzi Decoction polysaccharides enhanced the driving force for Ca2+ influx by increasing the content of polyamines, activating potassium channel, inducing cell membrane potential hyperpolarization.(4) Effect of Sijunzi Decoction polysaccharides on polyamine mediating calcium signaling pathways during cell migration: ①Sijunzi Decoction polysaccharides caused a promotion of [Ca2+]cyl, and reversed low level of [Ca2+]cyt induced by DFMO. ②Sijunzi Decoction polysaccharides caused an increase of calcium channel protein TRPC1 mRNA and protein expression levels, and recovered low levels of TRPC1 mRNA and protein expression induced by DFMO to normal levels.③Sijunzi Decoction polysaccharides caused a promotion of PLC-γ1 mRNA and protein expression and enhanced the content of IP3 which regulating intracellular calcium store releasing, reversed low level of IP3 and PLC-γ1 mRNA and protein expression caused by DFMO.④Cell migration were inhibited significantly by no calcium medium which removing the extracellular Ca2+, and the effect of Sijunzi Decoction polysaccharides on cell migration cultured by no calcium medium is less effective than by normal medium. These findings indicated that Sijunzi Decoction polysaccharides regulated [Ca2+]cyt by two ways, including promoting extracellular Ca2+ influx and intracellular Ca2+ store releasing, and the effect of former way is more significant.(5) Effect of Sijunzi Decoction polysaccharides on downstream targets of polyamines mediated calcium signaling pathway during cell migration:①Sijunzi Decoction polysaccharides caused a promotion of RhoA, Racl, Cdc42 protein expression levels, and reversed low levels of RhoA, Racl, Cdc42 protein expression caused by DFMO. In addition, Sijunzi Decoction polysaccharides increased Rho GTPase active state GTP bound RhoA, Racl, Cdc42 protein expression levels. ②Sijunzi Decoction polysaccharides increased cytoskeletal protein myosin Ⅱ protein expression level, and reversed low level of myosin II protein expression induced by DFMO. These findings indicated that Sijunzi Decoction polysaccharides regulated cytoskeleton reconstruction resulting in cell migration by regulating downstream targets of calcium signaling pathway, including Rho-GTPase and myosin Ⅱ.ConelusionPolysaccharides is one of the main effective components of Sijunzi Decoction, which promote intestinal epithelial cell migration. Sijunzi Decoction polysaccharides promote IEC-6 cell migration by regulating polyamines signaling pathway, and calcium signaling pathway is one of key factors in polyamines signaling pathway. Present research provide a reference for investing the underlying mechanism of Sijunzi Decoction on protecting gastrointestinal mucosa. |
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