Study On The Mechanism Of Ilex Rotunda Thunb,A Traditional Chinese Medicine In South Of The Five Ridges,In Repairing Gastrointestinal Mucosal Injury

Objective:To observe the preventive and therapeutic effects of Ilex Rotunda Thunb,a traditional Chinese medicine in south of the Five Ridges for regulating spleen and stomach,on model rats with gastric mucosal injury induced by ethanol,gastric mucosal injury by indomethacin and on gastrointestinal mucosal injury induced by indomethacin.To explore its effect and mechanism on the repair of gastrointestinal mucosal injury from the perspective of gastrointestinal mucosal barrier function and regulatory proteins of gastrointestinal function.Methods:1.Preparation of aqueous extract of Ilex Rotunda Thunb(ILTA):Ilex Rotunda Thunb were immersed in water for 1 hour,boiled for 2 hours,filtered with gauze and added pure water to boil for another 2 hours.Filter and combine the liquid medicine.At last,rotary evaporation concentration and freeze-drying it.2.The gastric mucosal injury model induced by ethanol in rats:male SD rats were randomly divided into control group,model group,ILTA 8 g·kg-1 and ILTA 16 g·kg-1 groups.After fasting for 24 hours,rats in control group and model group were given water by gavage,and rats in test drug group were given corresponding dose of ILTA by gavage.2 hours later,rats in model group and test drug group were given 5 ml·kg-1 ethanol by gavage.The rats in the control group were given water by gavage,and the animals were killed one hour later.3.The gastric mucosal injury model induced by indomethacin in rats:rats were randomly divided into control group,model group,ILTA 10 g·kg-1 and ILTA 20 g·kg-1 groups.After fasting for 24 hours,the control group and model group were given water by gavage,and the test drug group was given ILTA by gavage.An hour later,the model group and test drug group were given 60mg·kg-1 indomethacin by subcutaneous injection.The rats in the control group were injected with physiological saline subcutaneously,and the animals were killed 7 hours later.4.The small intestinal mucosal injury model induced by indomethacin in rats:the rats were randomly divided into normal group,model group,ILTA 8 g·kg-1 and ILTA 16 g·kg-1 groups.The control group and model group were given water by gavage,and test drug group was given ILTA in relative dose by gavage.An hour later,the model group and tests drugs group was injected subcutaneously 5mg·kg-1 indomethacin,and the control group was injected subcutaneously physiological saline for 4 days.On the fourth day,the animals were fasted.On the fifth day,the animals were killed.5.The expression of proteins ZO-1,occludin,claudin-1,E-cadherin,α-Catenin,β－Catenin,MUC1,MUC5AC,CDX-2 and Sox2 were detected by immunohistochemistry and Western blot6.The concentration of D-lactic acid in plasma was detected by ELISA.Result:1.ILTA has preventive and therapeutic effects on the model of gastrointestinal mucosal injury in rats:①The effect of gastric mucosa injury induced by ethanol in rats:lLTA 16 g·kg-1 group could prevent and treat the gastric mucosal injury induced by ethanol in rats(its gross score and pathological score decreased,compared with the model group,p<0.05);②The effect of gastric mucosa injury induced by indomethacin in rats:the gross score of ILTA 10 g·kg-1group was significantly lower than model group(p<0.05);③ The effect of small intestinal mucosa injury induced by indomethacin in rats:The gross score of 8 g·kg-1 group and 16 g·kg-1 group was significantly lower than that of model group(p<0.05).2.ILTA could promote tight junction protein expression in gastrointestinal mucosa of model rats through IHC or WB method:①The results of gastric mucosal damage induced by ethanol:ILTA(16 g·kg-1 or 8g·kg-1)can promote the expression of ZO-1,Occludin,Claudin-1,E-cadherin、α-catenin、β-catenin,compared with model group(p<0.05).②The results of intestinal mucosal damage induced by indomethacin:The expressions of ZO1,Occldin,Claudin-1,E-cadherin、α-catenin、β-catenin in gastric mucosa of ILTA(10 g·kg-1 or 20 g·kg-1)group were higher than those in the model group(P<0.05).③The results of intestinal mucosal damage induced by indomethacin:ILTA(16 g·kg-1 or 8g·kg1)can promote the expression of ZO-1,Occludin,Claudin-1,E-cadherin、α-catenin、βcatenin compared with model group(p<0.05).It is suggested that the IL TA can increase the expression of cell junction proteins proteins ZO-1,occludin,claudin-1,E-cadherin、αcatenin、β-catenin in gastrointestinal mucosa of rats induced by ethanol or indomethacin.3.ILTA promotes MUC1 and MUC5AC expression in gastric mucosa injury by IHC and WB methods:①The results of ethanol induced gastric mucosal damage:MUC1 and MUC5AC of gastric mucosa in the ILTA group was higher than that of model group(p<0.05);②The results of indomethacin induced gastric mucosal damage:MUC1 and MUC5AC protein of ILTA were increased compared with the model group(p<0.05).It is suggested that the ILTA can increase the expression of mucin proteins in gastric mucosa of rats induced by ethanol or indomethacin.4.ILTA promotes the expression of transcription factor in gastrointestinal mucosa injury by IHC and WB methods:①The results of ethanol induced gastric mucosal damage:Sox2 of gastric mucosa in the ILTA group was higher than that of model group(p<0.05);② The results of indomethacin induced intestinal mucosal damage:ILTA could promote the expression of CDX-2 in model rats,compared with model group(p<0.05),which suggested that the ILTA can increase the expression of Sox2 in gastric mucosa of rats induced by ethanol and CDX-2 in intestinal mucosa of rats induced by indomethacin.5.ILTA promotes the plasma D-lactate in intestinal mucosa injury induced indomethacin:ILTA could reduce the level of plasma D-lactate compared with the model group(p<0.05),which suggested that ILTA can improve the intestinal permeability damage.Conclusion:ILTA can prevent gastrointestinal mucosal injury induced by ethanol/indomethacin through increasing the expression of tight junction proteins,adherens junction proteins,protein MUC1,MUC5AC,Sox2 and CDX-2,decreasing intestinal permeability(Dlactate),promoting the recovery of gastrointestinal barrier function,thereby promoting the repair of damage to the gastrointestinal mucosa.The results enriches the understanding of the mechanism of action of traditional Chinese medicine in south of the Five Ridges with opsonizing spleen and stomach functions,and provides a new attempt to explore the mechanism of gastrointestinal protective effects of Chinese medicine in the south of the Five Ridges.