Study On The Synergistic Effect Of Berberine And Thymosin β4 On Gastric Ulcer Inflammation

Thymosin β4 is a small molecular weight protein with pharmacological effects such as anti-inflammatory,mucosal repair,and participation in cell apoptosis.Berberine is an effective ingredient isolated from Rhizoma Coptidis.It has the advantages of reducing inflammation,antibacterial,and participating in cell apoptosis.Domestic and foreign studies have shown that both thymosin β4 and berberine have anti-inflammatory effects,but their anti-inflammatory effects have certain limitations.Therefore,this experiment intends to further explore the synergistic effect of thymosin β4 and berberine in inhibiting inflammation.This research is divided into four parts,(1)The process optimization and preparation of thymosin β4,which included three parts:1)Explore the influence of single factors such as induction temperature,time,and inducer concentration on the expression of thymosin β4,2)Multi-factor orthogonal experiment verifies the effects of induction temperature,time and inducer concentration on the expression of thymosin β4,3)The recombinant Thymosin β4 protein was expressed,isolated and purified in prokaryotic Escherichia coli;(2)The berberine nanoparticles were prepared by ion cross-linking method,and they were characterized by the standard curve drawing,the influence of the different drug and carrier ratios on the particle size,optical phenomenon,stability,and in vitro release;3)Cytotoxicity test,flow cytometry,and ELISA inflammatory factor detection methods were used to evaluate thymosin β4 and berberine in vitro;4)The protective effect of ethanol-induced acute gastric ulcer in rats was studied by morphological observation and HE staining.Research results confirm(1)Thymosin β4 was treated with 0.25 mmol/L IPTG for 4 hours at 37℃,shaking bacteria for 4 hours,the protein expression was the best,and the concentration was 3.5 mg/mL.(2)The average particle size of berberine nanoparticles is 184.8±5.8 nm,the polydispersity(PDI)value is 0.17±0.02,and the zeta potential is+23.3±1.8 mV;the cumulative release within 6 hours is(70.52±2.91)%;Within 30 days,the particle size changes little,and there is no aggregation phenomenon;its encapsulation efficiency and drug loading are 90.91%and 8.26%,respectively.(3)It was confirmed by CCK-8 experiment and apoptosis experiment that 2μg/mL thymosin β4 and 5 μg/mL berberine are less cytotoxic to gastric mucosal epithelial cells,inhibiting cell apoptosis and synergistic effect better than the single-drug group;According to the results of the ELISA experiment,the concentration of IL-6 in the synergy group was 174.0±5.8 pg/mL,the concentration of IL-8 was 153.0±3.2 pg/mL,and the concentration of TNF-α was 327.4±1.8 pg/mL,which was lower than that of the thymosin β4 group and the berberine group,relatively;the IL-4 concentration of the synergy group was 27.9±0.6 pg/mL,which was higher than that of the thymosin β4 group and the berberine group.(4)In the experiments on the protective effects of ethanol-induced gastric ulcer in rats,through pathological tissue slices,it was found that thymosin β4 and berberine can reduce the degree of gastric mucosal damage,and the degree of damage after synergy is smaller.The above experiments show that thymosin β4 and berberine have a certain protective effect on ethanol-induced gastric ulcers,reduce inflammation,and have a better effect after synergy.