| Gastrointestinal mucosal injury is one of the common lesions of spleen deficiency syndrome.YiqiJianpi Chinese medicine can improve gastrointestinal lesions in patients with spleen deficiency syndromeand promote the repair of gastrointestinal mucosal damage by acting on polyamines.The research team found that the extracts of Codonopsispilosula,Astragalus membranaceus,Licorice and Sijunzi Decoction(polysaccharides,flavonoids)can promote the migration of small intestinal crypt stem cells(IEC-6),the mechanism is related to the regulation of Ca2+via the polyamine signaling pathway.70%alcohol elution fractions of Astragalus(total saponins>50%)can promote cell proliferation,Epithelial cell proliferation,migration,differentiation,adhesion and adhesion are important links in the repair of gastrointestinal mucosal injury,and one of the mechanisms of gastrointestinal mucosal protection of YiqiJianpi Chinese medicine.Objective:To observe the effects of total saponins of Astragalus and total saponins of Panax ginseng on the proliferation of intestinal epithelial cells and its regulatory proteins,eell cycle,eell migration and adhesion to connective proteins,and to observe the effects of Astragalus and Radix Ginseng on the intestinal mucosal injury induced by indomethacin in rats.The impact of the study,in order to explore the role of YiqiJianpi Chinese medicine Huangqi and ginseng gastrointestinal mucosal injury repair.Method:1.The extraction of Astragalus and Ginsenoside Saponins:Astragalus and Ginseng Decoction pieces were extracted by heating under reflux,n-butanol extraction,n-butanol site was passed through D101 macroporous resin;xanthine n-butanol fraction was collected by 50%and 70%ethanol.The liquid was freeze-dried to obtain total saponins of Astragalus;the ginseng n-butanol fraction was collected by 80%ethanol elution site and freeze-dried to obtain total ginsenosides.The main components of the extract of Astragalus membranaceus and total ginseng ginseng were analyzed by evaporative light high performance liquid chromatography(ELSD-HPLC).2.The effects of Astragalus and Ginsenosides on cell proliferation:Astragalus and ginseng total saponins(50 mg·L-1 or 100 mg·L-1 or 200 mg·L-1,the same below)can promote cell proliferation for 48 hours.It can reverse the inhibition of cell proliferation by DFMO(polyamine synthesis inhibitor).It suggests that it has the effect of promoting cell proliferation.3.The effect of aqueous extract of Astragalus and Radix Ginseng on rat intestinal mucosal injury:Indomethacin 6mg·kg-1·d-1 was injected subcutaneously for 4 days,and water extract of Astragalus membranaceus(5g·kg-1,15g·kg-1)or ginseng water extract(5g·kg-1,15g·kg-1,experimental dose according to the amount of crude drug)for 7d,8d killed animals,small intestinal mucosa,record gross intestinal mucosal damage,HE Staining was performed for pathological scoring.Results:1.Astragalus and ginseng total saponin components detection:The extraction rate of total saponins of Astragalus was 1.32%,the content of saponins was 88.8%.The main components of total Astragalus saponins are astragaloside A.The extraction rate of total ginseng saponins was 2.12%,the content of saponins was 92.2%,and the main three components are ginsenoside Rgl,ginsenoside Re and ginsenoside Rbl.2.The effects of Astragalus and ginseng total saponins of on cell proliferation:Astragalus and ginseng saponins(somg·L-1 or 100mg·L-1 or 200mg·L-1,the same below)could promote cell proliferation 48 hours after starvation.At the same time,Astragalus membranaceus and ginseng saponins could reverse the inhibitory effect of DFMO on cell proliferation.3.Theeffects of total saponins of Astragalus and ginseng on cell cycle:Compared with the normalgroup,the percentage of G0/G1 phase decreased significantly in Astragalus and ginseng total saponins(50mg·L-1or 100mg·L-1)group 48 hours after administration,while the percentage of G2/M+Sphase increased significantly.It can reverse the increase of G0/G1 percentage caused by DFMO and the decrease of G2/M+S phase percentage,which makes cells stay at rest and promotes the transition from G1 phase to S phase.4.Theeffects of total saponins of Astragalus and ginseng on the expression of proliferative related protein:Astragalus and ginseng total saponinsinereased the expression of c-Myc(transcription factor),RhoA(RhoGTPase)and Cdk2(cell cycle-dependent kinase)protein,and reversed the inhibition of DFMO on the expression of c-Myc,RhoA and Cdk2 protein.It suggests that promoting cell proliferation is associated with increased expression of proliferating regulatory proteins.5.Theeffects of total saponins of Astragalus and ginseng on cell migration:The total saponins of Astragalus and ginseng had no significant effect on cell migration.6.Theeffects of total saponins of Astragalus and ginseng on the expression of adhesion junction-related protein:The effects of the test drugs on the expression of related proteins were observed under the three culture conditions in the scratch damage cell migration model.? The effect of normal unloading on adhesion connexin protein expression:Astragalus and ginseng total saponins can increase the expression of transcription factor c-Mycprotein.Total saponins of Astragalus membranaceus can increase the expression of adhesion-associated protein E-cadherin,but has no significant effect on the expression of ?-catenin and ?-catenin.Ginseng total ginsenosides can increase the expression of E-cadherin and a-catenin,but have no significant effect on the expression of ?-catenin.?The effect of calcium free load on adhesion connexin protein expression:Astragalus and ginseng total saponins can increase the expression of transcription factor c-Mycprotein.The expression of adhesion-associated protein E-cadherin and a-catenin decreased with calcium-free culture,while Astragalus and ginseng total saponins had no significant effect on E-cadherin,a-catenin and p-catenin.? The effect of DFMO load on adhesion connexin protein expression:DFMO can decrease the expression of E-cadherin,?-cetenin and c-Myc,but has no significant effect on the expression of ?-catenin.Astragalus and ginseng saponins can reverse the inhibition of E-cadherin and c-Myc protein expression by DFMO,but The expression of ?-catenin and ?-catenin protein had no significant effect.Astragalus and ginseng total saponins can increase the expression of c-Myc protein under the three culture conditions,which indicates that the effect on c-Myc is one of the important effects.It provides a reference for further exploring the different pharmacological effects of total saponins of Astragalus membranaceus and ginseng from the perspective of regulating c-Myc,but its effect on the expression of adhesive connexin protein is weak.7.The effects of water extracts of astragalus and ginseng on intestinal mucosal injury induced by indomethacin in rats:Water extracts of Astragalus membranaceus(159·kg-1)and ginseng(5g·kg-1,15g·kg-1,)had preventive and therapeutic effects on intestinal mucosal injury in model rats,and the gross and pathological scores were all P<0.05 compared with model group;water extracts of Astragalus membranaceus(15g·kg-1)could reduce the mortality of model rats(P<0.05 compared with model group).?These results suggest that Astragalus membranaceus and ginseng can prevent and treat intestinal mucosal injury,and lay a foundation for the follow-up observation of the effects of Astragalus membranaceus and ginseng saponins at animal level.Conclusion:Total saponins of Astragalus membranaceus and ginseng can promote the proliferation of IEC-6 cells.The mechanism of action is related to the influence of cell cycle and the increase of the expression of cell proliferation regulatory proteins c-Myc,RhoA and Cdk2.They can also increase the expression of c-Myc protein in cell migration model,suggesting that the effect of Astragalus membranaceus and ginseng total sapolins on transcription factor c-Myc may be one of the synergistic effects of Astragalus membranaceus and ginseng total saponins on regulating the different effects of cells.Astragalus and ginseng total saponins have relatively weak effects on promoting cell migration,and the effect of enhancing the expression of adhesion junction protein in this model is also weak.The water extracts of Astragalus and ginseng have preventive and therapeutic effects on the intestinal mucosal injury induced by indomethacin in rats.This study provides a reference for exploring the repairing effects of Astragalus membranaceus and ginseng on gast:rointestinal mucosal injury from different angles. |
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