Reversal Of GES-1 Cell Damage And Its Decoction Method Based On Rhubarb Huanglian Xiexin Decoction

Dahang Huanglian Xiexin Decoction(DHXD)comes from the clause NO.154 of Treatise on Febrile Diseases.The clause says that Decoction of Radix et Rhizoma Rhei and Rhizoma Coptidis Xiexin suits the syndrome with the following symptoms and signs:A Vital-energy Stagnancy is taking shape at the epigastrium,soft when presse,and with floating pause under middle finger.Decoction of Radix et Rhizoma Rhei and Rhizoma Coptidis Xiexin:Radix et Rhizoma Rhei 2 liang,Rhizoma Coptidis 1 laing.Soak the above herbs an two sheng of mafeitang(boiling water with numerous bubbles on surface)for a while.Then filter the decoction.Take it in two doses.The optimal soaking condition of DHXD has not yet been confirmed,and no experimental research on the scientific intension of the "mafeitang" and "for a while" has been carried out.However,it is obvious that these two key conditions of soaking are closely related to the temperature of water and the time of soaking.Objective:By observing the effects of DHXD on the proliferation of GES-1 cells induced by ethanol in vitro,on the secretion of IL-4,VEGF,IL-8 and TNF-? in the cell media at the best timing,and on the expression of 3 key factors in NF-?B signaling pathway which include NF-?B,I?B? and IKK?,we discuss the mechanism of DHXD in repairing cellular damage and explore its best decoction method.Our study may provide new ideas to investigations about special decoction preparation in Treatise on Febrile Diseases.Method:(1)55 SD male rats in a SPF grade were randomly divided into 11 groups,including the normal control group(normal saline,20ml/(kg·d)),the DHXD A-? group(20 ml/(kg·d)and the Ranitidine group(20 ml/(kg·d).5 rats for each group.The rats were given drugs for 3 days consecutively.At 2 hours after the last administration,the rats were anesthetized with subcutaneous injection by of 0.3ml/100g 10%Chloral hydrate.After the rats were completely anesthetized,opened the abdomen and collected blood by abdominal aortic method.Took the upper serum from centrifuge(3000rpm,l0min).Mixed each group's upper serum,and got 11 groups of upper serum.(2)The GES-1 cells with good growth status were randomly divided into 12 groups as follows:blank group,model group,positive drug group,and drug A-? group(the specific condition of A-? group were shown in the Experimental Part).The concentrations of serum in the DMEM high glucose culture fluid were all 10%.After 24 hours of culture with 10%complete culture medium,the cells of each group were cultured with medicated serum for another 24 hours,and were then damaged by 6%ethanol to build a injury model.(3)The selection of best injury model and the effect of DHXD on the activity of damaged cells were evaluated by MTT detecting the inhibitory effect of the medicated serum on cell proliferation.(4)The concentrations of IL-4,VEGF,IL-8 and TNF-a in upper serum of each group were detected with enzyme linked immunosorbent assay(ELISA).(5)The protein expression of NF-?B,I?B? and IKK? were examined using Western-blot.Results:DHXD has a significant effect on the proliferation of GES-1 cells damaged by ethanol.MTT results showed that different concentrations of ethanol can cause damage to GES-1 cells,and the inhibition rate of 6%ethanol on cells for 3 hours reached 40%,indicating that 6%ethanol intervention for 3 hours was the best cell injury model.Effects of drugs on the viability of ethanol-injured cells:compared with the blank group,the activity of the cells in the model group,the drugs A,C,F,G,H,and I groups was significantly decreased(P<0.01 or P<0.05);Compared with the model group,the activity of B,D,E and positive drugs increased significantly(P<0.01 or P<0.05).It was indicated that the serum containing DHXD could reduce the damage of GES-1 cells to ethanol;the results of ELISA showed that:Compared with the blank group,the IL-4 content in the supernatant of the model group was significantly lower(P<0.01),and the VEGF content was significantly increased(P<0.01).Compared with the model group,the levels of IL-4 and VEGF increased in different degrees of drug groups(P<0.01).Among them,the effects of drug group B and E were slightly higher than those in other groups,but the difference between drug groups B and E was not significantly Obvious(P>0.05).Compared with the blank group,the content of IL-8?TNF-?in cell supernatants of model group are markedly increased(P<0.01),and compared with model group,the content of IL-8?TNF-?in drug groups are reduced in different extent(P<0.01 or P<0.05).The content of IL-8 in high concentration drug group's(100 ?,15 min)decline was significantly lower than other drug groups.And the content of TNF-a's descent degree is most obvious in drug group F(85 ?,48 min).the result of Western blot showed that compared with the blank group,NF-?B?I?B? and IKK? protein expression are significantly higher(P<0.01),the cell survival rate significantly reduced in the model group,and cell surviva.Conclusion:(1)DHXD has protective effect on gastric epithelial cell GES-1 in vitro.(2)DHXD protects gastric mucosa by regulating the protein expression of NF-?B?I?B? and IKK?,the key factors in the NF-?B signal pathway,and by mediating cytokine expression including IL-4,VEGF,IL-8 and TNF-?.(3)From the mechanism of DHXD in repairing cellular damage,it is suggested that the best temperature of "mafeitang" is 85?,and "for a while" is 15 minutes.