| Objective: Optimize the processing technology and formula of bitter wine soup,and explore the efficacy and mechanism of bitter wine soup on chronic pharyngitis through in vivo and in vitro experiments.Methods: 1.Optimization of bitter wine soup formula: take the average diameter of the bacteriostasis circle of Streptococcus hemolyticus A as the evaluation index of bitter wine soup formula optimization,and use single factor combined with orthogonal test to optimize the bitter wine soup formula.2.In vitro experiment: prepare drug containing serum of rats in blank group,positive drug group,low,medium and high dose group of Bitter wine soup and ancient recipe group of Bitter wine soup,and culture human bronchial epithelial cells(16HBE)in vitro 20 ug/ml lipopolysaccharide(LPS)and 10% medicated serum were used for intervention.CCK-8 was used to detect the effect of medicated serum in each group on the proliferation activity of 16 HBE cells induced by LPS,and ELISA was used to detect tumor necrosis factor in cell supernatant α(TNF-α)、Interleukin 6(IL-6)and interleukin-1β(IL-1β)The content of P65 and IκBα m RNA expression I was detected by PCR,Western Blot was used to detect the expression of P-p65 protein,and flow cytometry was used to detect the apoptosis of 16 HBE cells in other groups except Bitter wine soup Gufang group.3.In vivo experiment: 72 Wistar rats were randomly divided into blank group,model group,positive drug group and low,medium and high dose Bitter wine soup groups.The rat chronic pharyngitis model was prepared with 2.5% ammonia water.The dose of positive drug group was 1.3125 g·kg-1 daily,and the dose of low,medium and high dose groups was 0.5,1 and 2 m L·kg-1 daily respectively.The blank group and model group were given the same amount of normal saline,and each group was given by gavage once a day,Detection of TNF-α、IL-6 and IL-1β in rat serum by ELISA.HE staining was used to observe the morphological changes of rat pharynx mucosa,and Western Blot was used to detect p-65,P-p65,IκBα、P-IκBα、IKKα、P-IKKα Protein expression,PCR detection of P65 and IKK α m RNA expression.Results: 1.Preparation results of bitter wine soup ice cream: The best formula of bitter wine soup is: 60 m L of rice vinegar,30 g of egg white,10 g of clear Pinellia ternate,1 g of Phoenix Cloth.2.In vitro experiment results: Compared with the blank group,the proliferation activity of 16 HBE in the model group decreased(P<0.01),TNF-α、IL-6 and IL-1 β The content increased(P<0.01),the expression of P65 m RNA was up-regulated,IκBα m RNA expression was down regulated(P<0.01),P-p65 protein expression was up regulated(P<0.01),and apoptosis rate was increased(P<0.01).Compared with the model group,the proliferation activity of cells in the middle,high dose and ancient prescription groups of Bitter wine soup increased(P<0.01),TNF-α、IL-6、IL-1 β The content decreased(P<0.01),the expression of P65 m RNA was down regulated,IκBα m RNA expression was up regulated(P<0.01),P-p65 protein expression was down regulated(P<0.01),and apoptosis rate was down regulated(P<0.01).Compared with the ancient formula group of Bitter wine soup,the high dose group of Bitter wine soup has TNF-α、IL-1β The content decreased(P<0.01),and the expression of P65 m RNA decreased(P<0.01).3.In vivo experiment results: compared with the blank group,the model group TNF-α、IL-6 and IL-1β content was increased(P<0.01),the mucosa was keratinized obviously,and a lot of inflammatory cells were infiltrated,P-p65、P-IκBα、P-IKKα up regulation of protein expression(P<0.01),P65、IKKα m RNA expression was up-regulated(P<0.01).Compared with the model group,the medium and high dose group of Bitter wine soup had TNF-α、IL-6 and IL-1β The content decreased(P<0.01),the mucosal layer was in good shape,no keratinized stratified squamous epithelium was found,a small amount of inflammatory cells infiltrated,P-p65,P-IκBα,P-IKKα Protein expression was down regulated(P<0.01),P65,IKKα m RNA expression was down regulated(P<0.01).Conclusion: The Bitter wine soup after optimization of the process is easy to prepare and the dosage is clear.Its drug containing serum can improve the LPS induced inflammation damage of 16 HBE cells,and its efficacy is superior to the original formula.Bitter wine soup for P-p65,P-IκBα,P-IKKα the up-regulation of such indexes has obvious inhibitory effect,improves the morphology of rat pharyngeal mucosa,and down-regulates the level of inflammatory factors.Its mechanism may be related to the inhibition of NF-κB signal pathway activation. |
PDF Full Text Request