| Objective1.To clarify the status of Chinese and Western medicine studies of Helicobacter pylori（Hp）-associated atrophic gastritis（CAG）.2.The pathological status of gastric mucosa and the expression differences of Hh-Ptch-Smo-Gli and NOX/NF-κB/STAT1 signaling pathway were compared between HP-infected and non-HP-infected CAG patients to explore the biological mechanism of Hp promoting the transformation of CAG inflammatory cancer.3.A rat model of Hp-associated atrophic gastritis was established to observe the effects of Lian-Pu-Yin Jiaweifang on the expression of related cytokines in gastric mucosa and serum of the model rats,confirming that Lian-Pu-Yin Jiaweifang can regulate the core targets of Hh-Ptch-Smo-Gli and NOX/NF-κB/STAT1 signaling pathway to play a therapeutic role in Hp-associated atrophic gastritis.Methods1.Theoretical research refer to ancient books and relevant literature at home and abroad,sort out the historical development of the treatment of atrophic gastritis due to mechanical syndrome,sort out the epidemiological data and treatment status of HP-related CAG,and summarize the basic research of Lian-Pu-Yin Jiaweifang for treating HP-related CAG.2.Clinical observation Forty-three patients with CAG who met the criteria were enrolled and divided into CAG with Hp infection group（Hp+CAG group,n=21）and CAG without Hp infection group（HP-CAG group,n=22）.The histological changes of gastric mucosa of patients in the two groups were observed by hematoxylin-eosin（HE）staining.the relative expression levels of NOX1,NOX2,NOX4,STAT1,P65 and p-P65 in gastric mucosa were detected by Western Blot（WB）.Real-time fluorescence quantitative PCR（RT-q PCT）was used to detect the Expression of Gli1 m RNA,Gli2 m RNA,Gli3 m RNA,Shh m RNA,Smo m RNA,Ptch m RNA,NOX1 m RNA,NOX2 m RNA,NOX4 m RNA and NF-κB m RNA in gastric mucosa.3.Experimental study 60 SPF healthy male SD rats were randomly divided into blank group（n=10）and model group（n=50）.The rat model of HP-associated atrophic gastritis was established by multi-factor combination method.The rats were feed with a diet containing 0.03g·kg-1 ranitidine with 120mg·L-1 MNNG solution.After 2 days of feeding,they were fasted for 1 day and given 15%Na Cl 1m L /100 g intragastric administration at 56℃ on the first day,120 mg·L-1MNNG solution was given intragastric administration on the second day of feeding,and 40% 1m L /100 g ethanol was given to each animal on the day of fasting.By the end of the 6th week of continuous modeling,the mice were fazed for 12 h,and treated with antibiotics and indomethacin successively.The mice were given 2m L of 50g/L sodium bicarbonate solution every other day,and 1m L of Helicoba cter pylori suspension every 15 min,once a day,6 times in total.At the end of the 8th week,the multi-factor compound modeling method was continued in the modeling group until the end of the 16 th week.During the period,2 rats were randomly killed in each modeling group at 12,13,14,15 and 16 weeks to verify whether the modeling was successful.Nine rats died during the modeling process.At week 16,29 rats were divided into model group（n=9）,Vitacoenzyme group（n=10）and Lian-Pu-Yin Jiaweifang group（n=10）.Rats in Vitacoenzyme group were given Vitacoenzyme intragastric administration.Lian-Pu-Yin Jiaweifang decoction group was given Chinese herbal medicine.Intragastric dose of rats and adults（60kg）was converted,diluted with distilled water and stored in refrigerator at 4℃ for later use;Model group was given the same amount of normal saline intragastric administration,once a day,for 8 weeks.After the intervention,the gastric tissue and serum of rats were collected,and the gastric mucosal tissue morphology of rats in each group was observed by HE staining.Enzyme-linked immunosorbent assay（ELISA）was used to detect serum Interleukin-1β(IL-1β),Interleukin-6（IL-6）,Tumor necrosis factor-α（TNF-α）in rats,PepsinogenⅠ（PepsinogenⅠ,PGⅠ）,Pepsinogen Ⅱ（PepsinogenⅡ,PG Ⅱ）,Gastrin（GAS）;The relative expression levels of NOX1,NOX2,NOX4,STAT1,P65 and p-P65 in gastric mucosa were detected by WB.The expression of Gli1 m RNA,Gli2 m RNA,Gli3 m RNA,Shh m RNA,Smo m RNA,Ptch m RNA,NOX1 m RNA,NOX2 m RNA,NOX4 m RNA and NF-κB m RNA in gastric mucosa were detected by RT-q PCR.Results1.Theoretical research results1.1 Spleen and stomach weakness is the internal cause of CAG,Hp infection and diet disorder is the external cause,and dysregul-ation of qi is the basic pathogenesis of CAG.The disease is mainly characterized by deficiency of origin and excess of substance.Qi stagnation,damp-heat and blood stasis are common in the course of disease,and damp-heat induced blood stasis is the key pathogenesis in the development of gastric mucosal lesions.1.2 Hp,by means of virulence factors such as Cag A and Vac A,interferes with intracellular signaling pathways of host cells and intensifies gastric mucosa inflammation,atrophy and intestinal metaplasia by targeting different cellular proteins.Poor diet and lifestyle,such as smoking and alcohol consumption,increase the risk of intestinal metaplasia and carcinogenesis in CAG.Hp infec-tion,high-salt diet and the intake of N-nitroso compounds have a synergistic effect on the “inflammatory cancer transformation” in CAG.2.Clinical observation resultsHE staining results of gastric tissues in the two groups: Gastric mucosa in the Hp+CAG group showed necrosis and detachment of epithelial cells,unsmooth surface,reduced number and disordered arrangement of glands,metaplasia of intestinal epithelium,diffuse infiltration of lymphocytes and neutrophils in lamina proper.Gastric mucosa glands in the HP-CAG group were less in number and slightly disordered in arrangement,a small amount of intestinal metaplasia was observed locally,and lymphocyte and neutrophil infiltration in lamina propria was lighter than that in the Hp+CAG group.Rt-q PCR results: Compared with Hp-CAG group,the levels of Gli1 m RNA,Shh m RNA,Smo m RNA and Ptch m RNA in gastric mucosa of HP+CAG group were significantly decreased（P<0.01）.The levels of Gli2 m RNA,Gli3 m RNA,NOX1 m RNA,NOX2 m RNA,NOX4 m RNA and NF-κB m RNA were significantly increased（P<0.01）.WB detection results: Compared with Hp-CAG group,the relative expression levels of NOX1/GAPDH,NOX2/GAPDH,NOX4/GAPDH and p-P65/GAPDH in gastric mucosa of HP+CAG group were significantly increased（P<0.01）,and the relative expression levels of STAT1/GAPDH were significantly decreased（P<0.01）.There was no significant difference in the relative expression of P65/GAPDH between the two groups（P>0.05）.3.Experimental resultsGeneral conditions: rats in model group had less food intake,more wet,sticky or thin,smelly stools,hair become dry and dull,reaction and listless spirit;Lian-Pu-Yin Jiaweifang and Vitacoen zyme group: the mental state of the rats was better and the hair color was shiny,and the food intake was slightly more than the model group.Compared with the Vitacoenzyme group,the stools in the Lian-Pu-Yin Jiaweifang group were more normal and loose stools were less common.HE staining results of gastric tissue: The gastric tissue structure of rats in the blank group was normal,with clear muscularis,mucous membrane and submucosa,densely arranged glands and no intestinal metaplasia and inflammatory cell infiltration.The gastric tissue structure of rats in model group was severely abnormal,epithelial cells were necrotic and exfoliated,glands were extremely disordered,a large number of dysplasia occurred,some cancer foci were visible,intestinal metaplasia and inflame matory cell infiltration were obvious.The gastric tissue structure of the rats in the Vitacoenzyme group was slightly abnormal,including necrosis and shedding of epithelial cells,disordered arrangement of glands,a small amount of dysplasia and carcinoma,intestinal metaplasia and inflammatory cell infiltration.Prognosis The gastric tissue structure of Lian-Pu-Yin Jiaweifang group rats was close to normal,a small number of epithelial cells were disorganized and detached,the glands were arranged in order,some of them had mild atrophy,a small amount of intestinal metaplasia and inflammatory cell infiltration,and no abnormal hyperplasia or cancer tissue was observed.Serum ELISA results: Compared with blank group,serum levels of IL-1β,IL-6 and TNF-α in model group,Vitacoenzyme group and Lian-Pu-Yin Jiaweifang group were significantly increased（P<0.05）,while PGⅠ,PGⅡand GAS were significantly decreased（P<0.05）.Compared with model group,serum levels of IL-1β,IL-6 and TNF-αin Vitacoenzyme group and Lian-Pu-Yin Jiaweifang group were decreased,while PGⅠ,PGⅡand GAS were increased significantly（P<0.05）.PGR（PGⅠ/PGⅡ）in model group was significantly lower than that in blank group（P<0.05）,and PGR in Lian-Pu-Yin Jiaweifang group was higher than that in model group（P<0.05）.Compared with Vitacoenzyme group,the expression levels of IL-6 and TNF-αin serum of TCM group were decreased（P<0.01,P<0.05）,and the expression level of PGⅠwas increased（P<0.01）;There were no significant differences in the expression levels of IL-1β,PGⅡ,GAS and PGR in serum between Vitacoenzyme group and Lian-Pu-Yin Jiaweifang group（P>0.05）.RT-q PCR results of gastric tissue: Compared with blank group,the levels of Gli1 m RNA,Shh m RNA,Smo m RNA and Ptch m RNA in gastric mucosa of model group,Vitacoenzyme group and Lian-Pu-Yin Jiawei fang group were significantly decreased（P<0.01）.The m RNA levels of Gli2,Gli3,NOX1,NOX2,NOX4 and NF-κB were significantly increased（P<0.01）.Compared with model group,Gli1 m RNA,Shh m RNA,Smo m RNA and Ptch m RNA levels in gastric mucosa of rats in Vitacoenzyme group and Lian-Pu-Yin Jiaweifang group were increased（P<0.01）.The levels of Gli2 m RNA,Gli3 m RNA,NOX1 m RNA,NOX2 m RNA,NOX4 m RNA and NF-κB m RNA were significantly decreased（P<0.01）.Compared with Vitacoenzyme group,the levels of Gli1 m RNA,Shh m RNA and Smo m RNA in gastric mucosa of Lian-Pu-Yin Jiaweifang group were increased（P<0.05）.The expression levels of Gli2 m RNA,Gli3 m RNA,NOX1 m RNA,NOX2 m RNA,NOX4 m RNA and NF-κB m RNA were decreased（P<0.01）.There was no significant difference in the expression of Ptch m RNA in gastric mucosa between the Vitacoenzyme group and the Lian-Pu-Yin Jiaweifang group（P>0.05）.WB results: Compared with blank group,the relative expression levels of NOX1/GAPDH,NOX2/GAPDH,NOX4/GAPDH and p-P65 /GAPDH in gastric mucosa of model group,Vitacoenzyme group and Lian-Pu-Yin Jiaweifang group were significantly increased（P<0.01）,and the relative expression levels of STAT1/GAPDH were significantly decreased（P<0.01）.Compared with model group,the relative expression levels of NOX1/GAPDH,NOX2/GAPDH,NOX4/GAPDH and p-P65/GAPDH in gastric mucosa of rats in Vitacoenzyme group and Lian-Pu-Yin Jiaweifang group were significantly decreased（P<0.01）,while the relative expression levels of STAT1/GAPDH were obviously increased（P<0.05,P<0.01）.Compared with the Vitacoenzyme group,the relative expression levels of NOX1/GAPDH,NOX2/GAPDH,NOX4/GAPDH and p-P65 /GAPDH in gastric mucosa of Lian-Pu-Yin Jiaweifang group were significantly decreased（P<0.01）,while the relative expression levels of STAT1/GAPDH were obviously increased（P<0.01）.There was no significant difference in the relative expression of P65/GAPDH among all groups（P>0.05）.Conclusions1.Hp-related CAG is mainly characterized by dampness-heat syndrome of spleen and stomach.With the progression of the disease,gastric mucosal lesions are aggravated and blood stasis syndrome is common.Eradication of Hp,low-salt diet,proper intake of fresh fruits and vegetables,less spicy pickled food,smoking cessation and alcohol restriction and use Traditional Chinese medicine treatment based on syndrome differentiation are beneficial to delay HP-related CAG gastric mucosal lesions.2.Hp infection may lead to long-term inflammation of gastric mucosa,promote atrophy and intestinal metaplasia,and increase the risk of cancer by inhibiting Hh-Ptch-Smo-Gli signaling pathway and abnormal activation of NOX/NF-κB/STAT1 signaling pathway.3.Lian-Pu-Yin Jiaweifang reduced the expression of inflammatory cytokines in serum of Hp-associated precancerous lesion of gastric cancer model rats,increased the levels of GAS,PGⅠand PGⅡin serum,restored PGR,reactivated Hh-Ptch-Smo-Gli signaling pathway,and adjusted NOX/NF-κB/STAT1 pathway.Significantly improved the gastric tissue morphology of model rats,treated Hp-related atrophic gastritis,and played a role in preventing or blocking “inflammatory cancer transformation”. |
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