Protective Effects Of Huangqi-Gegen Herb Pair On Ethanol-induced Gastric Mucosal Injury

Research BackgroundIn recent years, the number and amount of consumption of alcohol drinking population increases every year, the incidence of gastric mucosal damage delicate wine diseases also increases. Alcohol-induced digestive system diseases including gastritis, gastric ulcer, duodenal ulcer, alcoholic liver disease, cirrhosis of the liver. Long-term alcohol consumption can induce intestinal metaplasia, intestinal metaplasia is excessive gastric mucosal lesions to a stage.The study of herb pair is an important part of the research on the traditional Chinese medicine, and a breakthrough for the study of the compound of Chinese medicine. Huangqi-Gegen herb pair on first appeared in books in the Qing Dynasty《Zheng zhi hui bu》 by LI Yongcui. Many medical exerpt think this herb pair has deinebriating action. Our previous in vivo study showing that, Astragalosides and Astragaloside Ⅳ as the main active site of Astragalus can reduce ethanol-induced gastric mucosal injury, protection of mucosal barrier, maintaining the integrity of the gastric mucosa structure. In vitro study showed Astragaloside and Astragalosides IV can reduce ethanol-induced apoptosis in gastric epithelial cells and promote gastric epithelial cells have been damaged to repair, and its mechanism of action Astragaloside and Astragalosides IV have relationship with Bcl-2/Bax singnal pathway. Therefore, the study takes Huangqi-Gegen herb pair as the breakthrough point to investigate the material basis and mechanism of protecting gastric mucosal damage.ObjectiveTo optimize the water extraction process of Huangqi-Gegen herb pair in protecting gastric mucosal injury by orthogonal test coupled with pharmacodynamics experiment. To Establishment the fingerprint of Huangqi-Gegen herb pair;the peak of the fingerprint associated with gastric mucosal injury score, using gray correlation method to determine the contribution of the composition in Huangqi-Gegen herb pair which against the gastric mucosal injury.Observe the different morphological changes which caused by Huangqi Gegen herb pair that against the gastric mucosal injury Study of apoptosis-related genes and proteins. To explore the mechanisms for the protection of gastric mucosal injury of Huangqi Gegen herb pairMethods1. To optimize the water extraction process of huangqi-gegen herb pair Gastric mucosal injury index was calculated to study the pharmacodymamic effects. With the content of puerarin as the observation index, and using the boiling time, boiling frequency, the amount of water as factors. The best condition of water extraction process of Huangqi-Gegen herb pair protecting gastric mucosal injury as follow:adding 10-fold water for 3 times, with 30 min per time.2. High performance liquid chromatography (HPLC) method to establish Huangqi-Gegen herb pair of different compatibility proportion(3:1、2:1、 1:1、1:2、1:3) of fingerprint. Making gray correlation analysis using the relative peak area of the fingerprint in different Huangqi-Gegen herb pair and the gastric mucosal damage index of mice. The contribution of the isoflavone chemical composition to the protection of the gastric mucosa was defined by the correlation degree.3. To observe the protective effect of Huangqi-Gegen herb pair on the gastric mucosa damage induced by ethanol in rats. The damage index of gastric mucosal in the visual lesion and the pathological sections of the stomach were calculated. The effect of Huangqi-Gegen herb pair on the morphology of gastric mucosal lesions was observed by HE staining.4. The effect of Huangqi-Gegen herb pair on the key genes and proteins of the apoptotic signaling pathway. Using the Real time PCR, Western-blot and immunohistochemistry to detecte the expression of Bcl-2/Bax/Caspase-3 gene and protein in the apoptotic signaling pathway.Results1.The optimal condition of water extraction process of huangqi-gege herb pair in protecting gastric mucosal injury was as follows:adding 10-fold water to decoct for 3 times,30 min for each time, its puerarin (15.90±0.04mg/ ml) higher than other traditional Chinese medicine group, the gastric mucosal injury score (14.84±11.82) lower than other traditional Chinese medicine group, the results show that this condition of boiling the decoction to protect acute gastric mucosal lesion much better than other groups.2. Establish an HPLC method of huangqi-gegen herb pair with different compatibility, determined eight components of the herb pair which are puerarin, daidzin, daidzein, genistein, dyes genistin, calycosin, calycosin glucoside; the Huangqi-Gegen herb pair of 3:1 compatibility group have more effective to protect the gastric mucosal injury; the contribute of eight components in the herb pair to protect gastric mucosal injury in the order of genistein>puerarin>daidzein>daidzein>Formononetin>calycosin>genistin>caly cosin glucoside3. Visually observed surface normal gastric mucosa of rats, no damage;the mucosa of rats in the model group have red color, there are cords or spotting; mild mucosal injury each treatment group, only a few spotting. HE staining showed normal gastric epithelial cells in intact rats, no cell loss, lamina propria glands intact;compared with the normal group, in model group the mucosal tissue destruction, loss of epithelial cells, necrosis, glandular disorders,submucosal edema, inflammatory cell infiltration, glandular dilatation, extravasation of red blood cells;positive group and the high dose group showed a small amount of epithelial cell loss, damage was lighter than the model group, middle dose group and low dose group showed epithelium partial loss, mucosal injury is slightly heavier than positive group and high dose group but are lighter than the model group.4. Compared with the* model group, Huangqi-Gegen herb pair can increase the mRNA expression of Bcl-2 gene, reduced the mRNA expression of Bax and Caspase-3 gene;Immunohistochemistry and Western-blot results showed that compared with the model group, Huangqi-Gegen herb pair can increase the protein expression levels of Bcl-2, reduced the protein expression levels of Bax and Caspase-3.Conclusion1. The best condition of water extraction process of Huangqi-Gegen herb pair protecting gastric mucosal injury as follow:adding 10-fold water for 3 times, with 30 min per time.2. Huangqi-Gegen herb pair has a protective effect on ethanol induced gastric mucosal injury, of which 3:1 compatibility group protection of gastric mucosal injury is better than that of other compatibility group; Of the 8 isoflavone compounds identified in the Huangqi-Gegen herb pair, the contribution of genistein in protecting gastric mucosa damage was higher than that of puerarin and other isoflavone compounds.3. Huangqi-Gegen herb pair can reduces ethanol induced gastric mucosal bleeding, edema, erosion, and other pathological injury. Its mechanism may be with the Huangqi-Gegen herb pair involved in the signal pathway of apoptosis, upregulation of anti apoptosis gene and protein expression, inhibition of Pro apoptotic gene and protein expression.