Traditional Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia

Abstract

The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia. The traditional Chinese medicine comprises the following components by weight: 15g of astragalus membranaceus, 20g of radix fici simplicissimae, 10g of bighead atractylodes rhizome, 20g of sculellaria barbata, 10g of curcuma zedoary, 10g of rhizoma sparganii, 15g of nacre mother of pearl and 10g of fructus aurantii. The traditional Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia is used for preparing traditional Chinese medicine preparations or traditional Chinese medicine decoction pieces for treating chronic atrophic gastritis with intestinal metaplasia. Animal experiments prove that the traditional Chinese medicine composition has an improvement effect on the body weight, gastric mucosa lesion, gastric digestion function and the like of mice with chronic atrophic gastritis and intestinal transformation, and the improvement effect is related to inhibition of Notch signal channels and promotion of cell apoptosis. Clinical experiments prove that the reverse rates of OLGA and OLGIM stages of a test group respectively reach 64.29% and 47.62%, which are obviously higher than those of a control group (Plt; 0.05) of the method.

Description

Technical Field

[0001] The invention belongs to the technical field of traditional Chinese medicines, and particularly relates to a traditional Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia. Background Art

[0002] "All diseases are caused by spleen deficiency", spleen and stomach weakness is the initiating factor of chronic atrophic gastritis, which runs through the whole process of the disease. Due to invasion of external evil, improper diet, or long-term illness, old age and other factors, spleen and stomach weakness, on the basis of spleen and stomach deficiency, or due to liver qi, qi movement disorder, resulting in spleen and stomach dysfunction, water and moisture dysfunction, or accumulation into phlegm, or endogenous heat, or long-term disease, gastric collateral blood stasis, qi and blood stagnation, condensation into poison, stasis and poison are intertwined, resulting in complex disease and lingering difficulty in cure. The transformation of chronic atrophic gastritis is a progressive pathological process, among which chronic non-atrophic gastritis is the beginning of the disease, the disease is in the qi, and the main pathogenesis is gastric qi stagnation; the "inflammation-cancer" transformation process is the development of the disease, from qi into blood, long-term disease into collaterals, from deficiency to excess, with gastric collateral stasis as the main pathogenesis, which is "initially qi stagnation in the meridians, and blood injury into the collaterals after a long time". The Golden Chamber states: "No evil is without poison. Heat is transformed from poison, change is caused by poison, and stasis is caused by poison." Stagnation and stasis can be transformed over time. On the contrary, poison damages the blood vessels and causes blood stasis, or poison stagnates the qi and causes blood stasis, or poison consumes yin fluid and causes blood stasis. Stagnation is the norm, and toxicity is the change. Stagnation turns to poison over time, and long-term toxicity causes stasis. Stagnation and toxicity are lingering and cause disease. Stasis and heat toxicity are both pathological products and new pathogenic factors. If they are not removed for a long time, they will promote atrophy of gastric mucosal glands, intestinal metaplasia, dysplasia and even cancer. The formation of stasis and toxicity is a process of quantitative change to qualitative change when qi, blood and dampness are stuck to each other to a certain extent. Its continuous evolution is the key to the transformation of chronic atrophic gastritis from "inflammation to cancer".

[0003] Based on the fact that chronic atrophic gastritis "inflammation-cancer" is transformed into a chronic process, its main pathogenesis, syndrome differentiation and treatment, and prognosis are quite different. The research team divides it into early and progressive stages in terms of treatment. In the early stage, spleen and stomach weakness is the main symptom, accompanied by pathological factors such as qi stagnation, damp heat, and blood stasis. The treatment is mainly to strengthen the spleen and replenish qi, and to regulate qi and regulate the middle, clear away heat and detoxify, or activate blood circulation and remove blood stasis. In the progressive stage, spleen and stomach weakness, phlegm and blood stasis, and gastric collateral stasis are the main pathogenesis, and strengthening the body and calming the middle, eliminating phlegm and dispersing knots, and removing blood stasis and attacking toxins are the main treatment methods.

[0004] In addition, at present, modern medical treatment of gastric cancer (GC) is mainly based on causal treatment (eradication of Helicobacter pylori), symptomatic treatment and follow-up monitoring. Although eradication of Helicobacter pylori is the main drug treatment method for improving the pathological state of CAG in modern medicine, studies have shown that it cannot effectively reduce the risk of moderate to severe CAG patients developing GC.

[0005] CAG is a disease with advantages in TCM. TCM has the advantages of overall regulation, improvement of clinical symptoms and mucosal pathological conditions in the treatment of CAG. Multiple systematic reviews have confirmed the effectiveness and safety of TCM in the treatment of CAG. Starting from the "Eighth Five-Year Plan" scientific and technological breakthrough plan, the research team has been committed to the clinical and basic research of CAG for more than 30 years. It proposed that "spleen deficiency and stomach collateral stasis" is the key pathogenesis of CAG and created a series of treatment prescriptions.

[0006] Chronic atrophic gastritis (CAG) is a common intractable disease of the digestive system, characterized by a decrease in the number of glands in the lamina propria of the gastric mucosal epithelium, with or without intestinal adenomas or pseudopyloric adenomas.

[16] CAG with moderate to severe intestinal metaplasia and / or intraepithelial neoplasia is closely related to the occurrence of gastric cancer and is an important precancerous lesion of gastric cancer. Western medicine treatment for CAG has not yet made a breakthrough. There are limited types of drugs for preventing and reversing CAG clinically, and current drugs focus more on relieving patients' symptoms.

[0007] A large number of studies have confirmed that traditional Chinese medicine has significant potential in reversing CAG and its associated intestinal metaplasia and intraepithelial neoplasia. The research team has summarized its etiology and pathogenesis in long-term clinical practice. We believe that spleen deficiency is the basis of CAG, and "qi stagnation, dampness obstruction, and blood stasis" are the symptoms of the disease. Therefore, it is proposed that "spleen deficiency is the root, qi and blood dampness are the symptoms, and blood stasis is the change" is the main pathological characteristics of CAG, and the basic treatment method is to strengthen the spleen and clear heat, regulate qi and activate blood circulation.

[0008] The invention patent "A Chinese medicine composition, its preparation and application" applied by the research team on November 29, 2019, includes 15-40 parts of Atractylodes macrocephala, 0-30 parts of Radix Pseudostellariae, 0-30 parts of Poria cocos, 5-15 parts of Fructus Aurantii Immaturus, 0-30 parts of Perilla stem, 0-30 parts of Cortex Cervi Paniculatae, 15-30 parts of Scutellaria barbata, 10-30 parts of Curcuma zedoariae, 15-40 parts of Astragalus, 15-45 parts of Prunus armeniacae, 0-30 parts of Fritillaria thunbergii and 0-20 parts of Eupolyphaga sinensis. The Chinese medicine composition not only effectively relieves clinical symptoms and improves gastric mucosal lesions, but also reverses gastric precancerous lesions, blocks the process of carcinogenesis, and fundamentally reduces the incidence and mortality of gastric cancer. However, considering the characteristics of the long course of the disease and the need for long-term medication, Eupolyphaga sinensis is slightly toxic and is not recommended for long-term or excessive use. In addition, the price is relatively expensive, and the economic burden of medication is high for patients. The research team simplified and optimized the prescription. Clinical observations from the Guangdong Province Traditional Chinese Medicine Strengthening Project in 2015 showed that the use of Curcuma zedoaria alone had a weak effect on improving intestinal metaplasia, and most patients with this disease had physical and mental symptoms such as poor sleep quality. Summary of the invention

[0009] In order to overcome the problems existing in the prior art, the addition of Trillium has a more obvious effect on improving intestinal metaplasia; patients with this disease are prone to physical and mental symptoms such as poor sleep quality; adding mother-of-pearl has been found in clinical observations to further improve patients' physical and mental symptoms such as sleep. Based on clinical syndrome differentiation and addition and efficacy observation, and the consideration of "special disease special prescription", a new prescription for the treatment of chronic atrophic gastritis with intestinal metaplasia was finally formed: 15g of Astragalus, 20g of Prunus armeniaca, 10g of Atractylodes macrocephala, 20g of Scutellaria barbata, 10g of Curcuma, 10g of Trillium, 15g of Mother-of-pearl, and 10g of Citrus aurantium.

[0010] To achieve the above objectives, the technical solution adopted in the first aspect of the present invention is:

[0011] A Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia, comprising:

[0012] Astragalus, quinquefolius, Atractylodes macrocephala, Scutellaria barbata, Curcuma zedoaria, Trillium, mother of pearl, Citrus aurantium.

[0013] Preferably, by weight, the formula is:

[0014] 15g Astragalus, 20g Five-fingered Peach, 10g Atractylodes, 20g Scutellaria barbata, 10g Curcuma, 10g Trillium, 15g Mother of Pearl, 10g Citrus aurantium.

[0015] The technical solution adopted in the second aspect of the present invention is:

[0016] The Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia as described above is used for preparing Chinese medicine preparations or Chinese medicine pieces or Chinese medicine freeze-dried powder for treating chronic atrophic gastritis with intestinal metaplasia.

[0017] The technical solution adopted in the third aspect of the present invention is:

[0018] A method for preparing the above-mentioned lyophilized powder of traditional Chinese medicine for treating chronic atrophic gastritis with intestinal metaplasia comprises:

[0019] Step 1: Weigh 10-30 parts of Astragalus, 15-30 parts of Prunus mume, 10-30 parts of Atractylodes macrocephala, 10-30 parts of Scutellaria barbata, 5-20 parts of Curcuma, 5-20 parts of Trigonella ternata, 5-30 parts of Mother of Pearl and 5-20 parts of Fructus Aurantii Immaturus respectively;

[0020] Step 2: Mix and crush the astragalus, quinquefolius, atractylodes, scutellaria barbata, zedoaria, trillium, mother-of-pearl and fructus aurantii weighed in step 1 to obtain crushed drugs, and then sieve to obtain coarse drug powder;

[0021] Step 3: Soak the crude drug powder obtained in step 2 in water for 1 to 2 hours, boil it over high heat, and then decoct it over low heat for 1 to 2 hours, filter and separate it to obtain a filter residue and a primary filtrate;

[0022] Step 4: Add water to the filter residue obtained in step 3, boil for 1 to 2 hours, filter and separate to obtain a secondary filtrate, and combine the secondary filtrate with the primary filtrate to obtain a water extract;

[0023] Step 5: The water extract obtained in step 4 is concentrated under reduced pressure at 60-65° C. to obtain concentrated medicine, and then freeze-dried in a vacuum to obtain lyophilized powder of traditional Chinese medicine.

[0024] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects or advantages:

[0025] The Chinese medicine composition of the present invention has an improving effect on the body weight, gastric mucosal lesions, gastric digestive function and the like of mice with chronic atrophic gastritis and intestinal metaplasia, and the improving effect is related to inhibiting the Notch signaling pathway and promoting cell apoptosis.

[0026] The reversal rates of OLGA and OLGIM in the experimental group were 64.29% and 47.62%, respectively, significantly higher than those in the control group (P<0.05). It not only effectively improved the pathological state of CAG patients, but also significantly improved the quality of life scores of patients in the physiological (clinical manifestations), independence and psychological fields. Especially in terms of mental health, patients showed a more positive emotional state, significantly alleviated anxiety symptoms and better psychological adaptability. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0028] In the attached picture:

[0029] Figure 1 This is the weight change trend chart of mice in each group from 12 to 24 weeks. Figure 1 In the data, Control is the blank group; Model is the model group; YSP is the folic acid group; JQHF-L is the low-dose Chinese medicine group; JQHF-M is the medium-dose Chinese medicine group; JQHF-H is the high-dose Chinese medicine group.

[0030] Figure 2 The body weight changes of mice in each group at 12, 16, 20, and 24 weeks. Figure 2 A: weight of mice in each group at 12 weeks; B: weight of mice in each group at 16 weeks; C: weight of mice in each group at 20 weeks; D: weight of mice in each group at 24 weeks. Compared with the blank group, *** P < 0.001, ** P<0.01; compared with the model group, ### P < 0.001, ## P<0.01, # P<0.05.

[0031] Figure 3 This is the trend chart of food intake changes in each group of mice from 12 to 24 weeks. Figure 3 In the data, Control is the blank group; Model is the model group; YSP is the folic acid group; JQHF-L is the low-dose Chinese medicine group; JQHF-M is the medium-dose Chinese medicine group; JQHF-H is the high-dose Chinese medicine group.

[0032] Figure 4 The changes in food intake of mice in each group at 12, 16, 20, and 24 weeks. Figure 4 A: food intake of mice in each group at 12 weeks; B: food intake of mice in each group at 16 weeks; C: food intake of mice in each group at 20 weeks; D: food intake of mice in each group at 24 weeks. Compared with the blank group, *** P < 0.001, ** P<0.01, * P<0.05; compared with the model group, ### P < 0.001, ## P<0.01.

[0033] Figure 5 This is a graph showing the changing trend of drinking water in each group of mice from week 12 to 24.

[0034] Figure 6 The changes in drinking water of mice in each group at 12, 16, 20, and 24 weeks. Figure 6 A: water intake of mice in each group at 12 weeks; B: water intake of mice in each group at 16 weeks; C: water intake of mice in each group at 20 weeks; D: water intake of mice in each group at 24 weeks. Compared with the blank group, *** P < 0.001, ** P<0.01, * P<0.05.

[0035] Figure 7 AB-PAS and HE staining of mice in each group (24 weeks), Fig. 9 In the data, Control is the blank group; Model is the model group; YSP is the folic acid group; JQHF-L is the low-dose Chinese medicine group; JQHF-M is the medium-dose Chinese medicine group; JQHF-H is the high-dose Chinese medicine group.

[0036] Figure 8 The expression level of D-xylose in serum of mice in each group, Fig. 9 In the experiment, Control was the blank group; Model was the model group; YSP was the folic acid group; JQHF-L was the low-dose Chinese medicine group; JQHF-M was the medium-dose Chinese medicine group; JQHF-H was the high-dose Chinese medicine group. *** P < 0.001, ** P<0.01; compared with the model group, #P<0.05. Blank group, model group, folic acid group, n=6 in each group; each dose group of Chinese medicine, n=7 in each group. Fig. 9 The expression levels of serum gastric digestive enzyme PGⅠ and PGⅡ and PGR in each group of mice. Fig. 9 A: serum gastric digestive enzyme PGⅠ expression level of mice in each group; B: serum gastric digestive enzyme PGⅡ expression level of mice in each group; C: PGR situation of mice in each group. Compared with the blank group, *** P < 0.001, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05. Blank group, model group, folic acid group, n=6 in each group; each dose group of Chinese medicine, n=7 in each group.

[0037] Fig.10 The expression levels of serum GAS, MTL and PGE2 in each group of mice. Fig.10 A: serum GAS expression level of mice in each group; B: serum MTL expression level of mice in each group; C: serum PGE2 expression level of mice in each group. Compared with the blank group, *** P < 0.001, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05. Blank group, model group, folic acid group, n=6 in each group; each dose group of Chinese medicine, n=7 in each group.

[0038] Fig.11 The expression levels of TNF-α, IL-6, and IL-1β in serum of mice in each group. Fig.11 A: serum TNF-α expression level of mice in each group; B: serum IL-6 expression level of mice in each group; C: serum IL-1β expression level of mice in each group. Compared with the blank group, *** P < 0.001, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05. Blank group, model group, folic acid group, n=6 in each group; each dose group of Chinese medicine, n=7 in each group.

[0039] Fig.12 The relative expression levels of CDX2, MUC2, KLF4, Villin1 and SOX2 mRNA in gastric mucosa of mice in each group. Fig.12Note: A: relative expression level of CDX2 mRNA in mouse gastric mucosa; B: relative expression level of MUC2 mRNA in mouse gastric mucosa; C: relative expression level of KLF4 mRNA in mouse gastric mucosa; D: relative expression level of Villin1 mRNA in mouse gastric mucosa; E: relative expression level of SOX2 mRNA in mouse gastric mucosa. Compared with the blank group, *** P < 0.001, ** P<0.01, * P<0.05; compared with the model group, ### P < 0.001, ## P<0.01, # P<0.05. Blank group, model group, folic acid group, n=6 in each group; each dose group of Chinese medicine, n=7 in each group.

[0040] Fig.13 Protein expression and localization of MUC2 and Villin1 in the gastric mucosa of mice in each group, protein expression of MUC2 and Villin1 in the gastric mucosal tissue of mice in each group (immunohistochemistry, 400×, scale bar 200 μm).

[0041] Fig.14 The relative expression levels of Notch1 and Hes1 mRNA in gastric mucosa of mice in each group, Fig.14 In the figure, A: relative expression level of Notch1 mRNA in mouse gastric mucosa; B: relative expression level of Hes1 mRNA in mouse gastric mucosa. Compared with the blank group, *** P < 0.001, ** P<0.01; compared with the model group, ## P<0.01, # P<0.05. Blank group, model group, folic acid group, n=6 in each group; each dose group of Chinese medicine, n=7 in each group.

[0042] Fig.15 The relative expression levels of Bax, Bcl2 and Caspase9 mRNA in gastric mucosa of mice in each group. Fig.15 In the figure, A: relative expression level of Bax mRNA in mouse gastric mucosa; B: relative expression level of Bcl2 mRNA in mouse gastric mucosa; C: relative expression level of Caspase9 mRNA in mouse gastric mucosa. Compared with the blank group, *** P<0.001; compared with the model group, ### P < 0.001, ## P<0.01, # P<0.05. Blank group, model group, folic acid group, n=6 in each group; each dose group of Chinese medicine, n=7 in each group.

[0043] Fig.16Protein expression and localization of Notch1, Hes1, Bax, and Bcl2 in gastric mucosa of mice in each group. Protein expression of Notch1, Hes1, Bax, and Bcl2 in gastric mucosa of mice in each group (immunohistochemistry, 400×, scale bar 200 μm).

[0044] Fig.17 The protein expression levels of Notch1, Caspase9, Bax, and Bcl2 in gastric mucosa of mice in each group. Fig.17 A: Western blotting to detect Notch1 protein expression in mouse gastric mucosa; B: Relative expression level of Notch1 protein in mouse gastric mucosa; C: Western blotting to detect Caspase9 protein expression in mouse gastric mucosa; D: Relative expression level of Caspase9 protein in mouse gastric mucosa; E: Western blotting to detect Bax and Bcl2 protein expression in mouse gastric mucosa; F: Optical density ratio of Bax / Bcl2 protein expression in mouse gastric mucosa. Compared with the blank group, ** P<0.01, * P<0.05; compared with the model group, ## P<0.01, # P<0.05. n=3 per group. DETAILED DESCRIPTION

[0045] The following is combined with Figure 1-Figure 17 The preferred embodiments of the present invention are described. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0046] The experimental methods and detection methods in the following embodiments are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

[0047] Example 1

[0048] A Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia, the composition is, by weight:

[0049] 15g Astragalus, 20g Five-fingered Peach, 10g Atractylodes, 20g Scutellaria barbata, 10g Curcuma, 10g Trillium, 15g Mother of Pearl, 10g Citrus aurantium.

[0050] The present invention is prepared by adding or subtracting the spleen-strengthening, heat-clearing and blood-activating prescription - Weiweiqing granules, which was previously studied by the research group. The invention mainly consists of astragalus, atractylodes, quinquefolius, scutellaria barbata, zedoaria, trillium, mother-of-pearl and fructus aurantii.

[0051] The main herb in the formula, Astragalus, is sweet in taste, slightly warm in nature, and enters the spleen and lung meridians. It can replenish the body's Qi, and has the effects of expelling toxins and promoting tissue regeneration, greatly replenishing the Qi of the lungs and spleen.

[0052] The assistant drug Atractylodes macrocephala is sweet and warm, and is an essential drug for replenishing Qi, strengthening the spleen, and eliminating bloating. It can strengthen the spleen and harmonize the middle, dry spleen dampness, and warm and invigorate spleen yang without damaging yin fluid.

[0053] Astragalus can nourish the spleen and stomach, and its function of detoxifying and promoting tissue regeneration hits the two major pathogenesis of CAG, "weak spleen and stomach, and internal accumulation of toxic evil". When combined with Atractylodes macrocephala, which can nourish qi and strengthen the spleen, it can assist the spleen in transporting and transforming qi and blood.

[0054] The assistant drug, Fructus Croton, is slowly invigorating the Qi without causing fire, replenishing the Qi without raising it, and strengthening the body without hindering the evil. Together with Astragalus and Atractylodes, it has the effect of strengthening the spleen and invigorating Qi. The monarch and the assistant are complementary to each other, which can not only enhance the function of invigorating the Qi and strengthening the spleen, but also restrain the dryness of Astragalus and Atractylodes. It is especially suitable for the characteristics of this disease with spleen deficiency causing dampness and long-term depression turning into heat.

[0055] The assistant drug Scutellaria barbata can clear away heat, detoxify, promote blood circulation and remove blood stasis, and can control the stasis and toxins in the stomach meridians.

[0056] The use of Trillium and Curcuma together can promote qi and resolve blood, eliminate accumulation and relieve pain, and regulate qi and blood circulation. They are the key medicines for resolving blood and eliminating symptoms. They can be used together with Astragalus, Atractylodes, and Eleutherodactyla to resolve blood stasis and toxins without damaging vital energy.

[0057] Mother of pearl softens and disperses lumps, reduces acidity and relieves pain, calms the nerves and calms convulsions, while Scutellaria barbata clears away heat, detoxifies and removes blood stasis, and both are adjuvants; Citrus aurantium is bitter, pungent, sour and warm in nature, and has the effects of regulating qi, relieving fullness, relieving stagnation and relieving bloating, making the whole prescription nourishing without obstruction, and is an adjuvant.

[0058] Patients with this disease suffer from indigestion, flatulence, acid reflux and other symptoms due to spleen and stomach dysfunction, which in turn affects sleep. The adjuvant mother of pearl has the effects of calming the liver and suppressing yang, calming the nerves and calming the nerves. It can be used to reduce acid and relieve pain, promote digestion, and thus improve stomach discomfort. It can also calm the nerves and improve sleep. The adjuvant Citrus aurantium has the effects of regulating qi and relieving fullness, promoting stagnation and relieving bloating, making the whole prescription nourishing without obstruction. The whole prescription is used together, and it is nourishing while clearing, and nourishing without stagnation, playing the effects of strengthening the spleen and regulating qi, clearing away heat and detoxifying, and activating blood circulation and unblocking collaterals, which fits the pathogenesis of chronic atrophic gastritis with intestinal metaplasia, "spleen deficiency, qi stagnation, heat stagnation and collateral stasis".

[0059] Embodiment 2:

[0060] The Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia as described in Example 1 is used for preparing a Chinese medicine preparation for treating chronic atrophic gastritis with intestinal metaplasia. The preparation is a granular preparation.

[0061] A method for preparing a traditional Chinese medicine preparation for treating chronic atrophic gastritis with intestinal metaplasia, comprising:

[0062] Step 1: Weigh 10-30 parts of Astragalus, 15-30 parts of Prunus mume, 10-30 parts of Atractylodes macrocephala, 10-30 parts of Scutellaria barbata, 5-20 parts of Curcuma, 5-20 parts of Trigonella ternata, 5-30 parts of Mother of Pearl and 5-20 parts of Fructus Aurantii Immaturus respectively;

[0063] Step 2: Mix and crush the astragalus, quinquefolius, atractylodes, scutellaria barbata, zedoaria, trillium, mother-of-pearl and fructus aurantii weighed in step 1 to obtain crushed drugs, and then sieve to obtain coarse drug powder;

[0064] Step 3: Soak the crude drug powder obtained in step 2 in water for 1 to 2 hours, boil it over high heat, and then decoct it over low heat for 1 to 2 hours, filter and separate it to obtain a residue and a primary filtrate;

[0065] Step 4: Add water to the filter residue obtained in step 3, boil for 1 to 2 hours, filter and separate to obtain a secondary filtrate, and combine the secondary filtrate with the primary filtrate to obtain a water extract;

[0066] Step 5: The water extract obtained in step 4 is concentrated under reduced pressure at 60-65° C. to obtain concentrated medicine, and then freeze-dried in a vacuum to obtain freeze-dried powder, i.e., the Chinese medicine composition.

[0067] 1. Animal Experimentation

[0068] 1. Study on the efficacy of Chinese herbal medicine combination in the treatment of chronic atrophic gastritis with intestinal metaplasia in mice

[0069] 1.1 Changes in body weight, food intake, and water intake of mice in each group

[0070] 1.1.1 Body weight changes of mice in each group

[0071] As shown in Table 1, during the intervention period, the body weight of mice in the blank group, folic acid group, and Chinese medicine group decreased at the initial stage of administration, began to increase at 14 weeks, and began to decrease at 20-21 weeks; while the overall trend of the body weight of mice in the model group was a gradual decrease. Figure 1 .

[0072] The weight changes of mice in each group were calculated at 12 weeks, 16 weeks, 20 weeks and 24 weeks. After normality test, the weight of mice at 12 weeks and 16 weeks were in accordance with normal distribution and homogeneous variance. The results of one-way analysis of variance showed that there were significant differences in weight among the groups (P<0.001). Pairwise comparison showed that compared with the blank group, the model group, folic acid group, low-dose Chinese medicine group, medium-dose Chinese medicine group and high-dose Chinese medicine group were statistically different (P<0.001). The weight of the blank group was significantly higher than that of the model group, folic acid group and each dose group of Chinese medicine; but there was no statistical difference among the model group, folic acid group and each Chinese medicine group (P>0.05). After normality test, the body weight of mice at week 20 was not completely in line with normal distribution (model group P<0.05). Kruskal-Wallis H test was used to compare the two groups. Compared with the blank group, the model group and folic acid group had statistical differences, and there was no statistical difference in the Chinese medicine groups; compared with the model group, the high-dose Chinese medicine group had statistical differences (P<0.05). The body weight of mice at week 24 was in line with normal distribution and homogeneous variance. One-way analysis of variance was used. The results showed that there were significant differences in body weight among the groups (P<0.001). Pairwise comparison showed that compared with the blank group, the model group, folic acid group, low-dose Chinese medicine group, medium-dose Chinese medicine group, and high-dose Chinese medicine group had statistical differences (P<0.001). The body weight of the blank group was significantly higher than that of the model group, folic acid group, and each dose group of Chinese medicine; compared with the model group, the low-dose Chinese medicine group, medium-dose Chinese medicine group, and high-dose Chinese medicine group had statistical differences. See Figure 2 .

[0073] 1.1.2 Changes in food intake of mice in each group

[0074] As shown in Table 2, during the intervention period, the food intake of mice in the blank group, model group, folic acid group, and Chinese medicine group first decreased, then increased, and then decreased; the food intake of the model group began to increase in the 15th week and gradually decreased in the 18th week; the food intake of the folic acid group began to increase in the 14th week, fluctuated and increased from the 20th week to the 22nd week, and began to decrease in the 22nd week; while the food intake of mice in the blank group and Chinese medicine groups began to increase in the 14th week and gradually decreased in the 21st week. Figure 3 .

[0075] The changes in food intake of mice in each group at 12 weeks, 16 weeks, 20 weeks and 24 weeks were calculated. After normality test, the food intake of mice in each group was in accordance with normal distribution and variance was equal at week 12. One-way analysis of variance was used, and the results showed that there was no significant difference in food intake among the groups (P = 0.05). At week 16, the food intake of mice in each group did not conform to normal distribution (P < 0.05 for the medium-dose group of Chinese medicine). Kruskal-Wallis H test was used, and the results of pairwise comparison showed that compared with the blank group, the model group, folic acid group, low-dose group of Chinese medicine, and medium-dose group of Chinese medicine were statistically different, but there was no statistical difference between the model group, folic acid group, and Chinese medicine groups. The food intake of the blank group was significantly higher than that of the model group, folic acid group, and each dose group of Chinese medicine. After normality test, at the 20th week, the food intake of mice in each group conformed to normal distribution and variance. The one-way ANOVA and pairwise comparison results showed that compared with the blank group, the model group, folic acid group, low-dose Chinese medicine group, medium-dose Chinese medicine group, and high-dose Chinese medicine group were statistically different (P<0.001); compared with the model group, the folic acid group and each Chinese medicine group were statistically different (P<0.001). After normality test, at the 24th week, the food intake of mice in each group did not conform to normal distribution (low-dose Chinese medicine group P<0.05). The Kruskal-Wallis H test and pairwise comparison results showed that compared with the blank group, the model group and folic acid group were statistically different, and there was no statistical difference among the Chinese medicine groups (P>0.05); compared with the model group, the high-dose Chinese medicine group was statistically different (P<0.01), while the folic acid group, low-dose Chinese medicine group, and medium-dose Chinese medicine group were not statistically different. See Figure 4 .

[0076] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred embodiments of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

[0077] Table 2 Changes in food intake of mice in each group at 2, 16, 20 and 24 weeks or M(P 25 , P 75 )]

[0078]

[0079] Note: Blank group, model group, folic acid group, n=6 in each group; low-, medium-, and high-dose Chinese medicine groups, n=7 in each group.

[0080] 1.1.3 Changes in drinking water of mice in each group

[0081] As shown in Table 3, during the intervention period, the water intake of mice in the blank group first decreased, then began to gradually increase in the 14th week, and then began to decrease in the 21st week; the water intake of mice in the model group first decreased, then began to increase in the 16th week, and then began to gradually decrease in the 18th week; the water intake of mice in the folic acid group, the low-dose Chinese medicine group, the medium-dose Chinese medicine group, and the high-dose Chinese medicine group first decreased after the intervention, then began to gradually increase in the 14th week, and then began to slowly decrease in the 20th week. Figure 5 .

[0082] The changes in water intake of mice in each group at 12 weeks, 16 weeks, 20 weeks and 24 weeks were calculated. After normality test, the water intake of mice in each group was in accordance with normal distribution and homogeneous variance at week 12. The one-way ANOVA showed significant differences among the groups (P<0.01). The results of pairwise comparison showed that compared with the blank group, the model group, folic acid group and each dose group of Chinese medicine were statistically different; but there was no statistical difference among the model group, folic acid group and each dose group of Chinese medicine (P>0.05). At week 16, the water intake of mice in each group did not conform to normal distribution (blank group P<0.05). The Kruskal-Wallis H test was used. The results of pairwise comparison showed that compared with the blank group, the low dose group of Chinese medicine had statistical differences (P<0.05), and there was no statistical difference among the other groups. The water intake of the blank group was significantly higher than that of the model group, folic acid group and each dose group of Chinese medicine. After normality test, at the 20th week, the amount of water consumed by mice in each group was in accordance with normal distribution and had equal variance. One-way ANOVA was used to compare the two groups, indicating that compared with the blank group, the model group, folic acid group, and Chinese medicine group were statistically different (P<0.01); but there was no statistical difference between the model group, folic acid group, and Chinese medicine group (P>0.05). After normality test, at the 24th week, the amount of water consumed by mice in each group was in accordance with normal distribution and had equal variance. One-way ANOVA was used to compare the two groups, indicating that compared with the blank group, the model group, folic acid group, and Chinese medicine group were statistically different, and there was no statistical difference between the other groups (P>005). See Figure 6 .

[0083] Table 3 Changes in water intake of mice in each group at 12, 16, 20, and 24 weeks or M(P 25 , P 75 )]

[0084]

[0085] Note: Blank group, model group, folic acid group, n=6 in each group; low-, medium-, and high-dose Chinese medicine groups, n=7 in each group.

[0086] 1.2. Pathological changes of gastric mucosa in mice of each group

[0087] Under AB-PAS staining, the pathological sections of the gastric mucosa of the mice in the blank group showed red staining of the mucosa layer, and no obvious blue staining was observed, which was the glands that secreted neutral mucus. The pathological sections of the gastric mucosa of the mice in the model group showed a large area of ​​blue staining, indicating the presence of intestinal metaplastic glands that secreted acidic mucus, indicating the presence of moderate to severe IM. Compared with the model group, the lesions in the folic acid group were alleviated, showing mild to moderate blue staining, indicating the presence of mild to moderate IM; most of the low-dose and medium-dose Chinese medicine groups showed mild blue staining, and a few showed moderate blue staining, indicating the presence of mild to moderate IM; most of the high-dose Chinese medicine groups had no obvious blue staining, and a few showed mild blue staining, indicating the presence of mild IM.

[0088] HE staining showed that the pathological sections of the gastric mucosa of mice in the blank group had neat cell structures, clear layers, regular and tight arrangement of epithelial cells, intact polarity of cells and nuclei, and some had dilated capillaries, showing mild inflammation. The pathological sections of the gastric mucosa of mice in the model group showed disordered glandular hierarchical structure, obvious thickening of the basement membrane, dilated capillaries, showing moderate inflammation, obvious vacuolar changes in cells, and mucus lakes of varying sizes formed in the glandular cavity, showing moderate to severe IM, most of which were severe intestinal metaplasia. Compared with the model group, the proportion of the lamina propria in the folic acid group was reduced, showing moderate atrophy, and some had capillary dilation, showing mild inflammation, cells showing vacuolar changes, and mucus lakes of varying sizes formed in the glandular cavity, showing mild-moderate IM, and most of them were moderate IM; the low-dose Chinese medicine group showed mild inflammation and mild-moderate IM; the medium-dose Chinese medicine group showed capillary dilation in some parts, showing mild inflammation, cells showing vacuolar changes, and mucus lakes of varying sizes formed in the glandular cavity, and most of them showed mild IM, and a few were moderate IM; the high-dose Chinese medicine group showed mild inflammation, and a few were mild IM. Figure 7 .

[0089] 1.3. Expression level of D-xylose in mouse serum

[0090] The expression level of D-xylose in serum of mice in each group was detected by ELISA.

[0091] As shown in Table 4, after normality test, the data of each group did not conform to normal distribution (partial P<0.05). The Kruskal-Wallis H test was used for pairwise comparison. The results showed that compared with the blank group, the model group and the folic acid group had statistical differences, and there was no statistical difference in the Chinese medicine groups (P>0.05). The expression level of D-xylose in the blank group was higher than that in the model group, the folic acid group and the Chinese medicine groups at different doses. Compared with the model group, the high-dose Chinese medicine group had statistical differences (P<0.05), and there was no statistical difference in the other intervention groups (P>0.05). Figure 8 .

[0092] Table 4 Serum D-xylose content of mice in each group [M (P 25 , P75 )]

[0093]

[0094] 1.4. Expression levels of gastric digestive zymogens PGⅠ and PGⅡ and PGR in mouse serum

[0095] The expression levels of serum gastric digestive enzyme PGⅠ and PGⅡ in each group of mice were detected by ELISA method, and the PGR (PGⅠ / PGⅡ ratio) of each group of mice was calculated. Fig. 9 .

[0096] As shown in Table 5, the PGI expression levels of the blank group, model group, folic acid group, low-dose Chinese medicine group, medium-dose Chinese medicine group and high-dose Chinese medicine group followed normal distribution and had equal variances. One-way ANOVA showed that there were significant differences in the expression levels of PGⅠ in each group (P<0.001). After pairwise comparison, the results showed that compared with the blank group, the model group and the folic acid group had statistical differences, and there were no statistical differences in the Chinese medicine groups (P>0.05). The PGⅠ expression level in the blank group was higher than that in the model group, folic acid group and each dose group of Chinese medicine; compared with the model group, the medium-dose Chinese medicine group and the high-dose Chinese medicine group had statistical differences, and there were no statistical differences in the folic acid group and the low-dose Chinese medicine group (P>0.05).

[0097] The PG II expression levels in the blank group, model group, folic acid group and each dose group of Chinese medicine followed normal distribution and had equal variance. One-way analysis of variance and pairwise comparison results showed that there was no statistical difference among the blank group, model group, folic acid group and each dose group of Chinese medicine (P>0.05).

[0098] Table 5 Serum PGI, PGII expression levels and PGR status of mice in each group

[0099]

[0100]

[0101] 1.5. Expression levels of gastrin, motilin, and prostaglandin E2 in mouse serum

[0102] The expression levels of serum gastrin (GAS), motilin (MTL) and prostaglandin E2 (PGE2) in each group of mice were detected by ELISA. Fig.10 .

[0103] As shown in Table 6, the GAS expression levels in the blank group, model group, folic acid group, and Chinese medicine dose groups obeyed normal distribution and had equal variances. One-way ANOVA showed that the GAS expression levels of the groups were significantly different (P<0.001). After pairwise comparison, the results showed that compared with the blank group, the model group and the folic acid group had statistical differences, and there were no statistical differences in the Chinese medicine groups (P>0.05). The GAS expression level in the blank group was lower than that in the model group, the folic acid group, and the Chinese medicine dose groups. Compared with the model group, the Chinese medicine medium dose group and the Chinese medicine high dose group had statistical differences, while the folic acid group and the Chinese medicine low dose group had no statistical differences (P>0.05).

[0104] The expression levels of MTL and PGE2 in each group followed normal distribution and had equal variances. One-way analysis of variance showed that there were significant differences in the expression levels of MTL and PGE2 in each group (P<0.01). After pairwise comparison, the results showed that compared with the blank group, there were statistical differences in the model group, folic acid group and low-dose Chinese medicine group, and there were no statistical differences in the medium-dose Chinese medicine group and high-dose Chinese medicine group (P>0.05). The expression levels of MTL and PGE2 in the blank group were higher than those in the model group, folic acid group and each dose group of Chinese medicine; compared with the model group, the high-dose Chinese medicine group had statistical differences (P<0.05), while there were no statistical differences in the folic acid group, low-dose Chinese medicine group and medium-dose Chinese medicine group (P>0.05).

[0105] Table 6 Expression levels of serum GAS, MTL and PGE2 in each group of mice

[0106]

[0107] 1.6. Expression levels of gastrin, motilin, and prostaglandin E2 in mouse serum

[0108] The expression levels of serum gastrin (GAS), motilin (MTL) and prostaglandin E2 (PGE2) in each group of mice were detected by ELISA. Fig.10 .

[0109] The GAS expression levels in the blank group, model group, folic acid group, and Chinese medicine dose groups followed normal distribution and had equal variances. One-way ANOVA showed that there were significant differences in the GAS expression levels among the groups (P<0.001). After pairwise comparison, the results showed that compared with the blank group, the model group and the folic acid group had statistical differences, and there were no statistical differences among the Chinese medicine groups (P>0.05). The GAS expression level in the blank group was lower than that in the model group, the folic acid group, and the Chinese medicine dose groups. Compared with the model group, the Chinese medicine medium dose group and the Chinese medicine high dose group had statistical differences, while the folic acid group and the Chinese medicine low dose group had no statistical differences (P>0.05).

[0110] The expression levels of MTL and PGE2 in each group followed normal distribution and had equal variances. One-way analysis of variance showed that there were significant differences in the expression levels of MTL and PGE2 in each group (P<0.01). After pairwise comparison, the results showed that compared with the blank group, there were statistical differences in the model group, folic acid group and low-dose Chinese medicine group, and there were no statistical differences in the medium-dose Chinese medicine group and high-dose Chinese medicine group (P>0.05). The expression levels of MTL and PGE2 in the blank group were higher than those in the model group, folic acid group and each dose group of Chinese medicine; compared with the model group, the high-dose Chinese medicine group had statistical differences (P<0.05), while there were no statistical differences in the folic acid group, low-dose Chinese medicine group and medium-dose Chinese medicine group (P>0.05).

[0111] 1.7. Expression levels of TNF-α, IL-6, and IL-1β in mouse serum

[0112] The expression levels of serum tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and interleukin 1β (IL-1β) in each group of mice were detected by ELISA. Fig.11 .

[0113] As shown in Table 7, the expression levels of TNF-α in the blank group, model group, folic acid group, and Chinese medicine dose groups obeyed normal distribution and had equal variances. One-way ANOVA showed that the expression levels of TNF-α in each group were different (P<0.001). After pairwise comparison, the results showed that compared with the blank group, the model group and folic acid group had statistical differences (P<0.001), and there were no statistical differences in the Chinese medicine dose groups (P>0.05). The expression level of TNF-α in the blank group was lower than that in the model group, folic acid group, and Chinese medicine dose groups. Compared with the model group, the high-dose Chinese medicine group had statistical differences (P<0.05), while the folic acid group, the low-dose Chinese medicine group, and the medium-dose Chinese medicine group had no statistical differences (P>0.05).

[0114] The IL-6 expression levels in each group followed normal distribution and had equal variances. One-way analysis of variance was used. After pairwise comparison, the results showed that compared with the blank group, the model group and the folic acid group had statistical differences, while there were no statistical differences among the various dose groups of Chinese medicine (P>0.05). The IL-6 expression level in the blank group was lower than that in the model group, the folic acid group and the various dose groups of Chinese medicine. Compared with the model group, the high-dose Chinese medicine group had statistical differences (P<0.05), while there were no statistical differences among the folic acid group, the low-dose Chinese medicine group and the medium-dose Chinese medicine group (P>0.05).

[0115] The expression levels of IL-1β in each group followed normal distribution and had equal variances. One-way analysis of variance was used. After pairwise comparison, the results showed that compared with the blank group, there were statistical differences in the model group and the folic acid group, but there were no statistical differences in the various doses of Chinese medicine groups (P>0.05). The expression level of IL-1β in the blank group was lower than that in the model group, the folic acid group and the various doses of Chinese medicine groups; compared with the model group, there was a statistical difference in the high-dose Chinese medicine group (P<0.01), while there was no statistical difference in the folic acid group, the low-dose Chinese medicine group and the medium-dose Chinese medicine group (P>0.05).

[0116] Table 7 Serum TNF-α, IL-6, and IL-1β expression levels of mice in each group

[0117]

[0118] 1.8. mRNA expression levels of intestinal metaplasia-related indicators in mouse gastric mucosa

[0119] As shown in Table 8, the mRNA expression levels of CDX2 in the gastric mucosa of mice in the blank group, model group, folic acid group, low-dose Chinese medicine group, medium-dose Chinese medicine group and high-dose Chinese medicine group followed normal distribution and homogeneous variance. One-way ANOVA showed that there were significant differences in the expression levels of CDX2 in each group (P<0.001). After pairwise comparison, the results showed that compared with the blank group, there were no statistical differences in the low-dose Chinese medicine group, medium-dose Chinese medicine group and high-dose Chinese medicine group (P>0.05), and there were statistical differences in the model group and folic acid group. The mRNA expression level of CDX2 in the gastric mucosa of mice in the model group was higher than that in the blank group. Compared with the model group, there were statistical differences in the medium-dose Chinese medicine group and the high-dose Chinese medicine group (P<0.01), while there were no statistical differences in the folic acid group and the low-dose Chinese medicine group (P>0.05).

[0120] The mRNA expression levels of MUC2 in the gastric mucosa of mice in each group followed normal distribution and homogeneous variance. One-way ANOVA showed that the expression levels of MUC2 in each group were significantly different (P<0.001). After pairwise comparison, the results showed that compared with the blank group, the high-dose Chinese medicine group had no statistical difference (P>0.05), and the model group, folic acid group, low-dose Chinese medicine group, and medium-dose Chinese medicine group had statistical differences. The mRNA expression level of MUC2 in the gastric mucosa of mice in the model group was higher than that in the blank group. Compared with the model group, the low-dose Chinese medicine group, medium-dose Chinese medicine group, and high-dose Chinese medicine group had statistical differences, and the folic acid group had no statistical difference (P>0.05).

[0121] The mRNA expression levels of KLF4 in the gastric mucosa of mice in each group followed normal distribution and had equal variances. One-way ANOVA showed that the expression levels of KLF4 in each group were significantly different (P<0.001). After pairwise comparison, the results showed that compared with the blank group, the high-dose Chinese medicine group had no statistical difference (P>0.05), and the model group, folic acid group, low-dose Chinese medicine group, and medium-dose Chinese medicine group had statistical differences. The mRNA expression level of KLF4 in the gastric mucosa of mice in the model group was higher than that in the blank group. Compared with the model group, the medium-dose Chinese medicine group and the high-dose Chinese medicine group had statistical differences, while the folic acid group and the low-dose Chinese medicine group had no statistical differences (P>0.05).

[0122] The mRNA expression levels of Villin1 in the gastric mucosa of mice in each group followed normal distribution and homogeneous variance. One-way ANOVA showed that the expression levels of Villin1 in each group were significantly different (P<0.001). After pairwise comparison, the results showed that compared with the blank group, all groups had statistical differences (P<0.001), and the mRNA expression level of Villin1 in the gastric mucosa of mice in the model group was higher than that in the blank group. Compared with the model group, the low-dose group, the medium-dose group, and the high-dose group of traditional Chinese medicine were statistically different (P<0.001), and there was no statistical difference in the folic acid group (P>0.05).

[0123] The mRNA expression levels of SOX2 in the gastric mucosa of mice in each group followed normal distribution and had equal variances. One-way ANOVA showed that there were significant differences in the expression levels of SOX2 in each group (P<0.001). After pairwise comparison, the results showed that compared with the blank group, all groups had statistical differences (P<0.001), and the mRNA expression level of SOX2 in the gastric mucosa of mice in the blank group was higher than that in the model group. Compared with the model group, there were statistical differences in the medium-dose Chinese medicine group and the high-dose Chinese medicine group, while there were no statistical differences in the folic acid group and the low-dose Chinese medicine group (P>0.05). Fig.12 .

[0124] Table 8 Relative mRNA expression levels of CDX2, MUC2, KLF4, Villin1 and SOX2 in gastric mucosa of mice in each group

[0125]

[0126] 1.9. MUC2 and Villin1 protein expression and localization

[0127] Immunohistochemistry was used to locate the protein expression of MUC2 and Villin1 in the gastric mucosa of each group of mice. Fig.13 .

[0128] The expression and localization of MUC2 protein were detected. The gastric mucosa of mice in the blank group was uniformly light yellow without obvious brown-yellow granules. The positive expression of protein in mice in the other groups was brown-yellow granules, mainly with yellow staining of the cytoplasm, some of which were darker and mostly located in the IM area. The model group was darker than the drug-treated group.

[0129] The expression and localization of Villin1 protein were detected. The positive expression of gastric mucosal protein in each group of mice was presented as brown-yellow granules, with the cytoplasm mainly yellow-stained, some of which were darker and mostly located in the IM area. The model group was darker than the blank group and the drug-treated group.

[0130] 2. Study on the mechanism of Chinese herbal medicine combination in treating chronic atrophic gastritis with intestinal metaplasia in mice

[0131] 2.1 Changes in the expression of Notch pathway and apoptosis-related mRNA levels in mouse gastric mucosa

[0132] As shown in Tables 9 and 10, the mRNA expression levels of Notch1 in the gastric mucosa of mice in the blank group, model group, folic acid group, low-dose Chinese medicine group, medium-dose Chinese medicine group and high-dose Chinese medicine group followed normal distribution and homogeneous variance. One-way ANOVA showed that the expression levels of Notch1 in each group were significantly different (P<0.001). After pairwise comparison, the results showed that compared with the blank group, all groups were statistically different (P<0.001), and the mRNA expression level of Notch1 in the gastric mucosa of mice in the model group was higher than that in the blank group. Compared with the model group, the medium-dose Chinese medicine group and the high-dose Chinese medicine group were statistically different, while the folic acid group and the low-dose Chinese medicine group were not statistically different (P>0.05).

[0133] The mRNA expression levels of Hes1 in the gastric mucosa of mice in each group followed normal distribution and had equal variances. One-way ANOVA showed that the expression levels of Hes1 in each group were significantly different (P<0.001). Pairwise comparisons showed that there were statistical differences in each group compared with the blank group, and the mRNA expression level of Hes1 in the gastric mucosa of mice in the model group was higher than that in the blank group. Compared with the model group, the high-dose Chinese medicine group had statistical differences (P<0.05), while there were no statistical differences in the folic acid group, the low-dose Chinese medicine group, and the medium-dose Chinese medicine group (P>0.05). Fig.14 .

[0134] The mRNA expression levels of Bax in the gastric mucosa of mice in each group followed normal distribution and homogeneous variance. One-way ANOVA showed that the expression levels of Bax in each group were significantly different (P<0.001). After pairwise comparison, the results showed that compared with the blank group, all groups had statistical differences (P<0.001), and the mRNA expression level of Bax in the gastric mucosa of mice in the blank group was higher than that in the model group. Compared with the model group, the low-dose group, medium-dose group, and high-dose group of traditional Chinese medicine were statistically different, and there was no statistical difference in the folic acid group (P>0.05).

[0135] The mRNA expression levels of gastric mucosal Bcl2 in each group of mice followed normal distribution and homogeneous variance. One-way ANOVA showed that the expression levels of Bcl2 in each group were significantly different (P<0.001). After pairwise comparison, the results showed that compared with the blank group, all groups had statistical differences (P<0.001), and the mRNA expression level of gastric mucosal Bcl2 in the model group was higher than that in the blank group. Compared with the model group, the folic acid group, the low-dose Chinese medicine group, the medium-dose Chinese medicine group, and the high-dose Chinese medicine group were all statistically different.

[0136] The mRNA expression levels of Caspase9 in the gastric mucosa of mice in each group followed normal distribution and had equal variances. One-way analysis of variance showed that there were significant differences in the expression levels of Caspase9 in each group (P<0.001). After pairwise comparison, the results showed that compared with the blank group, all groups had statistical differences (P<0.001), and the mRNA expression level of Caspase9 in the gastric mucosa of mice in the blank group was higher than that in the model group. Compared with the model group, the low-dose Chinese medicine group, the medium-dose Chinese medicine group, and the high-dose Chinese medicine group were statistically different, and there was no statistical difference in the folic acid group (P>0.05). Fig.15 .

[0137] Table 9 Relative expression levels of Notch1 and Hes1 mRNA in gastric mucosa of mice in each group

[0138]

[0139] Table 10 Relative mRNA expression levels of Bax, Bcl2 and Caspase9 in gastric mucosa of mice in each group

[0140]

[0141] 2.2 Expression and localization of Notch pathway and apoptosis-related proteins

[0142] Immunohistochemistry was used to detect and locate the protein expressions of Notch1, Hes1, Bax, and Bcl2 in the gastric mucosa of mice in each group. Fig.16 .

[0143] The expression and localization of Notch1 and Bcl2 proteins were detected. The gastric mucosa of mice in the blank group was uniformly light yellow without obvious brown-yellow granules. The positive expression of proteins in mice in the other groups was brown-yellow granules, mainly with yellow staining of the cytoplasm, some of which were darker and mostly located in the IM area. The coloration of the model group was darker than that of the drug-treated group.

[0144] The expression and localization of Bax protein were detected. The positive expression of gastric mucosal protein in each group of mice was presented as brown-yellow granules, mainly with yellow cytoplasm, some of which were darker and mostly located in the IM area. The coloration of the model group was lighter than that of the blank group and the drug-treated group, and the blank group was the darkest.

[0145] The expression and localization of Hes1 protein were detected. The positive expression of gastric mucosal protein in each group of mice was presented as brown-yellow granules, with the nucleus mainly stained yellow. Some parts were stained darker, mostly located in the IM area, while the model group was stained darker than the blank group and the drug-treated group.

[0146] 2.3 Expression levels of Notch pathway and apoptosis-related proteins

[0147] Western blotting was used to detect the expression levels of Notch pathway and apoptosis-related proteins. Fig.17 .

[0148] As shown in Table 11, the relative protein expression optical density values ​​of Notch1 in the gastric mucosa of mice in the blank group, model group, folic acid group, low-dose Chinese medicine group, medium-dose Chinese medicine group and high-dose Chinese medicine group followed normal distribution and homogeneous variance. One-way ANOVA showed that the Notch1 levels in each group were significantly different (P<0.01). After pairwise comparison, the results showed that compared with the blank group, the model group and folic acid group were statistically different, and the relative protein expression level of Notch1 in the gastric mucosa of mice in the model group was higher than that in the blank group. Compared with the model group, the high-dose Chinese medicine group was statistically different (P<0.01), while the folic acid group, low-dose Chinese medicine group and medium-dose Chinese medicine group were not statistically different (P>0.05).

[0149] The relative protein expression optical density values ​​of Caspase9 in gastric mucosa of mice in each group followed normal distribution and homogeneous variance. One-way ANOVA showed that there were significant differences in the levels of Caspase9 in each group (P<0.01). After pairwise comparison, the results showed that compared with the blank group, the model group had statistical differences (P<0.01), and the relative protein expression level of Caspase9 in gastric mucosa of mice in the blank group was higher than that in the model group. Compared with the model group, the low-dose Chinese medicine group had statistical differences (P<0.05), while there were no statistical differences in the folic acid group, the medium-dose Chinese medicine group and the high-dose Chinese medicine group (P>0.05).

[0150] The increase of Bax / Bcl2 ratio indicates the promotion of cell apoptosis. The ratio of optical density of Bax / Bcl2 protein expression in gastric mucosa of mice in each group obeyed normal distribution and had equal variance. One-way ANOVA showed that the ratio of Bax / Bcl2 in each group was significantly different (P<0.01). After pairwise comparison, the results showed that the model group had statistical difference compared with the blank group (P<0.01), and the ratio of gastric mucosa Bax / Bcl2 in blank group was higher than that in model group. Compared with the model group, the low-dose Chinese medicine group and the medium-dose Chinese medicine group had statistical difference, while the folic acid group and the high-dose Chinese medicine group had no statistical difference (P>0.05), and the ratios of the folic acid group and the Chinese medicine groups were higher than those of the model group.

[0151] Table 11 Relative average optical density levels of Notch1, Caspase9, and Bax / Bcl2 protein expression in gastric mucosa of mice in each group ( n=3)

[0152]

[0153] 3. Conclusion

[0154] 3.1 Successful construction of mouse model

[0155] N-methyl-N-nitrosourea (MNU), a DNA-directed alkylating agent, can exert its carcinogenicity without metabolic activation. This study used a multi-factor composite modeling method, including MNU and hunger and satiety disorder; hunger and satiety disorder simulates irregular eating in daily life, which can cause gastric acid secretion disorders and low human immune function, forming an immunosuppressive microenvironment in the stomach. As Chinese medicine says, long-term hunger and satiety disorder damages the spleen and stomach, and leads to spleen deficiency over time. At present, this method has been widely used in the construction of animal models of spleen deficiency syndrome. At the same time, MNU, a carcinogenic "poison", stimulates the gastric mucosa of mice for a long time and stays in the body for a long time, which can cause blood stasis. Blood stasis can turn into heat over time, forming the phenomenon of "heat stagnation and blood stasis", which manifests as gastric mucosal atrophy, intestinal metaplasia and dysplasia in the gastric mucosa. It is consistent with the description of the etiology and pathogenesis of chronic atrophic gastritis in Chinese medicine, and belongs to the disease-syndrome combination model of "weak spleen and stomach, heat stagnation and blood stasis", which is consistent with the treatment method of Chinese medicine combination used in the study.

[0156] At 12 weeks after modeling, the weight of mice in the model group was significantly reduced, and the food and water intake were reduced compared with the normal group. Under AB-PAS staining, the gastric mucosal pathology of mice in the model group showed blue staining, indicating the presence of intestinal metaplastic glands that secreted acidic mucus. HE staining showed that the mouse intestinal metaplasia model was successfully established.

[0157] 3.2 Therapeutic effect of Chinese medicine composition on CAG mouse model

[0158] At present, the Western medicine treatment of CAG is mainly to eradicate H. pylori. Apart from this, no drug has been clearly reported to reverse gastric mucosal lesions. Studies have suggested that certain vitamins and trace elements may help delay the progression of CAG, such as folic acid tablets and vitamin tablets. Folic acid tablets may reverse gastric mucosal lesions mainly by maintaining whole genome methylation. Therefore, this study used folic acid tablets as a control drug to evaluate the therapeutic effect of JQHF on CAG mice.

[0159] The study used mouse body weight, food intake, drinking water, pathological staining, serum D-xylose, serum pepsinogen, serum gastrointestinal hormones, serum inflammatory factors, and gastric mucosal intestinal metaplasia marker mRNA and protein expression levels as indicators to evaluate efficacy.

[0160] The experiment found that after 12 weeks of drug intervention, compared with the body weight, food intake and water intake of mice in the model group, the Chinese medicine combination group could increase the body weight, food intake and water intake of CAG mice with intestinal metaplasia to a certain extent.

[0161] Analysis of the pathological staining of the gastric mucosa of mice revealed that the glandular hierarchical structure of the mice in the model group was disordered, the basement membrane was significantly thickened, capillary dilation was visible, and the glandular cavity formed mucus lakes of varying sizes, which appeared as large areas of blue staining under AB-PAS staining, indicating the presence of intestinal metaplastic glands that secreted acidic mucus, showing moderate to severe IM. A few mice in the high-dose group of traditional Chinese medicine showed mild IM, and most showed mild inflammation. The low-dose group and the medium-dose group of traditional Chinese medicine had the second best effect, showing mild to moderate IM and mild inflammation. The folic acid group showed moderate atrophy, mild inflammation, mild to moderate IM, and most of them were moderate IM.

[0162] The serum D-xylose absorption rate is one of the microscopic indicators reflecting the animal model of spleen deficiency syndrome. The decrease in serum D-xylose content indicates that the small intestine absorption function is reduced in spleen deficiency syndrome. The experiment found that the serum D-xylose expression level of mice in the model group was significantly lower than that in the blank group, while that in the folic acid group and the Chinese medicine groups was higher than that in the model group, and the absorption function of the high-dose Chinese medicine group was better.

[0163] Serum pepsinogen, serum gastrointestinal hormones, and serum inflammatory factors can objectively reflect the inflammation, atrophy, and functional status of the gastric mucosa. The experiment found that the PGⅠ and PGR of the mice in the model group decreased, and GAS increased significantly. When the gastric body atrophied, PGⅠ and PGR decreased significantly. The reduction of gastric body acid glands would negatively feedback and lead to an increase in GAS. The results suggested that the gastric body mucosa of the mice in the model group may atrophy. The PGⅠ and PGR levels of the mice in each dose group of Chinese medicine were higher than those in the model group, and the GAS level was lower than that in the model group. However, the changes in the relevant indicators of the mice in the folic acid group were not obvious, indicating that the Chinese medicine composition may improve the atrophy of the gastric mucosa, and the effect is better than folic acid. In addition, it was found that JQHF can upregulate the expression levels of serum MTL and PGE2 in mice. The main physiological effects of MTL are to promote gastrointestinal motility, stimulate pepsin secretion, and induce regular gastrointestinal contraction. PGE2 can stimulate the production and secretion of gastric mucus, increase gastric mucosal blood flow, and maintain gastric mucosal blood microcirculation; suggesting that JQHF may improve gastric mucosal pathology by promoting gastrointestinal motility, blood circulation, pepsin and mucus secretion in mice. TNF-α is an inflammatory factor produced by activated monocytes or macrophages, which plays an important role in the occurrence and development of inflammation and tumors; TNF-α can induce DNA damage, inhibit DNA repair, and promote tumor cell growth; TNF-α may have other significant effects in the early stages of carcinogenesis, such as angiogenesis and invasion. IL-6 and IL-1β are interleukins (IL), and they are biological indicators of inflammatory response together with TNF-α. In terms of inflammatory factors, the expression levels of TNF-α, IL-6, and IL-1β in mice in the model group were increased, while the expression of inflammatory factors in the treatment group was decreased, indicating that JQHF and folic acid may have anti-inflammatory effects.

[0164] At present, it is believed that IM belongs to the intermediate stage of the gastric "inflammation-cancer" transformation model and is the further development of gastric atrophy. The risk of gastric cancer in patients with CAG and IM is higher than that in patients with simple atrophy. CDX2, MUC2, KLF4 and Villin1 are considered to be intestinal metaplasia markers; CDX2 and KLF4 are intestinal-specific transcription factors; the mucin encoded by the MUC2 gene is the main component of the colonic mucus layer; the villin encoded by Villin1 is the skeleton component of the striated border of intestinal epithelial cells. On the basis of AB-PAS and HE staining to determine the IM state of gastric mucosa, this study further detected the expression levels of intestinal metaplasia markers and SOX2; SOX2 is a transcription factor involved in gastric differentiation and negatively regulates intestinal differentiation. The experiment found that the mRNA expression level of intestinal metaplasia markers in the model group mice was upregulated compared with the normal group, and SOX2 was downregulated compared with the normal group. The immunohistochemical results showed that the protein expression of MUC2 and Villin1 in the model group was upregulated compared with the normal group, indicating that the gastric mucosal cells of the model group mice were transformed into intestinal mucosal cells. Under the treatment of JQHF or folic acid, the expression of intestinal metaplasia markers was downregulated and SOX2 was upregulated, indicating that JQHF and folic acid are effective in treating CAG with intestinal metaplasia, the high-dose Chinese medicine group had the best effect, and the Chinese medicine group had better effect than folic acid. JQHF and folic acid may be able to delay the progression of IM or even reverse IM.

[0165] In summary, it can be preliminarily concluded that the Chinese medicine composition has certain therapeutic effects in improving the weight loss and diet of CAG mice with intestinal metaplasia, improving the pathological state of the gastric mucosa of mice, and restoring their hormone and pepsinogen secretion levels, thereby treating CAG mice with intestinal metaplasia. The molecular mechanism of this therapeutic effect needs further experimental research.

[0166] 3.3 Effects of Chinese herbal medicine combination on the expression of Notch pathway and apoptosis-related molecules

[0167] The Notch signaling pathway is highly conserved in genetic evolution and is involved in mediating processes such as cell proliferation, apoptosis, and differentiation. Notch1 is one of the receptors of the Notch signaling pathway. Notch regulates many components of the tumor microenvironment, including immune cells as well as fibroblasts, endothelial cells, and mesenchymal cells. Its dysregulation leads to the development of various solid tumors. Studies have found that Notch1 is upregulated in clinical gastric cancer tissues. Compared with patients with low Notch1 levels, patients with poor differentiation and high Notch1 expression have a worse overall survival rate. The Notch signaling pathway is closely related to the occurrence and development of cancer. Hes1 belongs to the practice of helix-loop-helix transcription factors, which has the functions of regulating neural development, T lymphocyte development, and cancer occurrence. Hes1 is a downstream target gene of the Notch signaling pathway and is regulated by Notch1 signals. The normal pyloric glands and intestinal metaplasia glands in paracancerous tissues may reflect the regulatory role of Notch1 and Hes1 in the proliferation and differentiation of these glands. The experiment found that the mRNA expression level and protein level of Notch1 and Hes1 in the model group were significantly higher than those in the blank group, while those in the drug-treated group were lower than those in the model group; the protein expression level of Notch1 in the model group was higher than that in the blank group, while the protein expression levels of Notch1 in the folic acid group and the Chinese medicine groups were lower than those in the model group; all of these indicate that CAG with intestinal metaplasia is related to the Notch signaling pathway, and the Notch signaling pathway may be activated in the process of gastric mucosal tissue evolving from normal to intestinal metaplasia, while the Chinese medicine composition and folic acid may block the process of gastric mucosal deterioration by inhibiting the Notch signaling pathway.

[0168] Apoptosis is a highly regulated cell death process in animals. Under normal physiological conditions, apoptosis can help the body remove excess or damaged cells in time, maintain the homeostasis of the body's internal environment and the performance of normal physiological functions. Pro-apoptotic proteins (such as Bax and Bad) induce apoptosis by destroying the integrity of the mitochondrial membrane, leading to a decrease in MMP and biochemical changes inside and outside the mitochondrial membrane, promoting the release of Cyto C from mitochondria into the cytoplasm, activating Apaf-1, and forming apoptosomes together with Cyto C, Apaf-1 and pro-caspase-9 in the cytoplasm, and then activating Caspase-3 to induce apoptosis; on the contrary, anti-apoptotic proteins (such as Bcl-2 and Bcl-xl) effectively inhibit this process. Bax, as an important pro-apoptotic gene in the Bcl-2 family, can participate in regulating most of the apoptotic processes in animals. The expression level of Bax protein is proportional to the apoptotic ability of cells and can be used as an indicator for evaluating apoptosis; in contrast to Bax, Bcl-2 is an important anti-apoptotic gene in the Bcl-2 family, which is widely expressed in tumors and can significantly enhance the anti-apoptotic ability of tumors. Caspase is a family of aspartate-specific cysteine ​​proteases, which also play an important role in regulating apoptosis signaling pathways. Under the stimulation of damage-related molecular patterns such as D-gal, mitochondrial dysfunction occurs, which increases the release of Cyto C from mitochondria into the cytoplasm, promotes the formation of apoptotic bodies composed of cytoplasmic Cyto C, Apaf-1, and pro-caspase-9, and then cleaves pro-caspase-9 and pro-caspase-3 into cleaved-caspase-9 and cleaved-caspase-3 in sequence, further causing cell apoptosis; Caspase9 and Caspase-3 are considered to be another major cytopathological hallmark of the apoptotic process. RT-qPCR results showed that the mRNA expression levels of Bax and Caspase 9 in the model group were significantly lower than those in the blank group, while Bcl2 was higher than that in the blank group model, while the mRNA expression levels of Bax and Caspase9 in the folic acid group and each Chinese medicine group were higher than those in the model group, while Bcl2 was lower than that in the model group; WB results showed that the Bax / Bcl2 ratio and Caspase 9 protein level in the model group were significantly lower than those in the blank group, while those in the folic acid group and each Chinese medicine group were higher than those in the model group, and the increase in the Chinese medicine group was greater than that in the model group; Immunohistochemistry results showed that the expression level of Bax in the model group was significantly lower than that in the blank group, while Bcl2 was higher than that in the blank group model, while the mRNA expression levels of Bax in the folic acid group and each Chinese medicine group were higher than those in the model group, while Bcl2 was lower than that in the model group; It can be seen that the apoptosis mechanism of gastric mucosal cells in the model group mice was inhibited, while the apoptosis mechanism of gastric mucosal cells in the drug administration group was promoted. In summary, JPQF and folic acid may improve CAG with intestinal metaplasia by promoting cell apoptosis.

[0169] Animal experiments have shown that the Chinese medicine composition has an improving effect on the body weight, gastric mucosal lesions, gastric digestive function, etc. of mice with chronic atrophic gastritis with intestinal metaplasia, and this improvement is related to inhibiting the Notch signaling pathway and promoting cell apoptosis.

[0170] 2. Clinical Trials:

[0171] Experimental Design

[0172] A multicenter, double-blind, double-dummy, randomized, controlled, experimental clinical study design was adopted. This study was registered with ClinicalTrials.gov (No.NCT03658291) and approved by the medical ethics committees of three clinical centers: the First Affiliated Hospital of Guangzhou University of Chinese Medicine (approval number: ZYYECK

[2020] 008), Zhongshan Hospital of Traditional Chinese Medicine (approval number: ZSZY-2020-LL-306), and Hainan Provincial Hospital of Traditional Chinese Medicine (approval number: HNSZYY-2020-LL-018).

[0173] 1.1 Case source

[0174] The observed cases came from three domestic branch centers from January 2020 to December 2022, namely, the First Affiliated Hospital of Guangzhou University of Chinese Medicine, Hainan Provincial Hospital of Traditional Chinese Medicine, and Zhongshan Hospital of Traditional Chinese Medicine.

[0175] 1.2 Blind method

[0176] This study was blinded to the subjects, outcome assessors, and data analysts. The researchers concealed the treatment regimen of the subjects from the three, and the latter two did not participate in the drug treatment process, and independently evaluated and analyzed the outcomes of the subjects and their clinical data.

[0177] 1.3 Stratified block randomization

[0178] Using SAS9.2 statistical software, the number of clinical trial centers was used as the stratification factor, and the random number grouping table was generated according to the number of cases allocated and the random ratio of the sub-center. There were 3 clinical sub-centers in this project, and the experimental group and the control group were in a 1:1 ratio, and the random code was generated using the stratified block randomization method.

[0179] 2. Diagnostic criteria

[0180] 2.1 Western medicine diagnostic criteria

[0181] The diagnostic criteria were formulated with reference to the Consensus on Chronic Gastritis in China (2017, Shanghai) and the Consensus on the Diagnosis and Treatment of Chronic Atrophic Gastritis with Integrated Traditional Chinese and Western Medicine (2017) issued by the Chinese Society of Gastroenterology of the Chinese Medical Association: (1) Clinical symptoms: no symptoms; or common symptoms such as indigestion, upper abdominal discomfort, fullness, pain, etc.; or gastrointestinal symptoms such as loss of appetite, nausea, belching, acid reflux, nausea, bitter taste in the mouth, etc.; or systemic or mental symptoms such as fatigue, weight loss, forgetfulness, anxiety, depression, etc.; the above symptoms may be induced by improper diet, emotional excitement, depression, fatigue, weather changes, etc. (2) Endoscopy and gastric mucosal biopsy: ① The diagnosis by endoscopy is based on the red and white mucosa, with the white predominant color, the flattening or even disappearance of mucosal folds, the exposure of mucosal blood vessels, and the granular or nodular mucosa; if accompanied by bile reflux, erosion, intramucosal bleeding, etc., it is described as atrophic gastritis with bile reflux, erosion, intramucosal bleeding, etc.; ② In gastric mucosal biopsy, if the biopsy shows atrophy of the intrinsic glands, CAG can be diagnosed, regardless of the number and degree of atrophy of the biopsy specimen. However, biopsies should be avoided at lesions such as erosions.

[0182] 3. Inclusion criteria

[0183] ① Age 18-65 years old, regardless of gender; ② Chronic atrophic gastritis confirmed by gastroscopy and gastric mucosal pathological histological examination; ③ Helicobacter pylori test with negative result within the past month; ④ Normal verbal communication is possible; ⑤ Informed consent and voluntary participation.

[0184] 4. Exclusion criteria

[0185] ① Participated in other drug treatment research within the past month; ② Combined with autoimmune gastritis (type A chronic atrophic gastritis), peptic ulcer, gastroesophageal reflux disease, chronic esophagitis, hiatal hernia, hypertrophic gastritis, ulcerative colitis and other diseases; ③ High-grade gastric mucosal intraepithelial neoplasia, gastrointestinal mucosal lesions suspected of malignant transformation, gastrointestinal tumors; ④ Patients with malignant tumors who underwent surgery, radiotherapy, and chemotherapy within the past 5 years; ⑤ Patients with severe organic diseases that threaten their quality of life and are unable to cooperate with drug treatment. Such as: heart (NYH III-IV heart function, myocardial infarction with unstable hemodynamics, etc.), liver (decompensated cirrhosis, etc.), kidney (uremia stage of chronic renal failure, etc.), lung (tuberculosis, acute asthma attack, lung infection, etc.), autoimmune system (active systemic lupus erythematosus), etc.; ⑥ Those with allergic constitution or allergy to multiple drugs; ⑦ Those with severe mental illness who cannot cooperate with the researchers, such as schizophrenia, depression, anxiety, etc.; ⑧ Women who are pregnant, breastfeeding, or have fertility requirements; ⑨ Those who do not cooperate with the investigation.

[0186] 5. Elimination criteria

[0187] ① Those who do not meet the inclusion and exclusion criteria. ② Those whose missing items in the case report questionnaire reach more than 20%, and it is difficult to judge the efficacy. ③ Those who use intervention measures other than the protocol during the study. ④ Those who do not take medication as prescribed, such as: self-adjusting the type, dosage and usage of medication. The reasons for the exclusion of cases should be explained, and no statistical analysis of efficacy evaluation will be performed.

[0188] 6. Shedding criteria

[0189] ① Patients who have serious adverse events during the study and are not suitable to continue to participate in the trial; ② Patients with poor compliance and cannot strictly follow the treatment plan to complete the trial after enrollment; ③ Patients with missing clinical data and unable to conduct efficacy or safety evaluation; ④ Patients who are lost during the study. The reasons for dropouts should be explained, and some data will be included in the statistical analysis of efficacy evaluation.

[0190] 7. Interventions

[0191] Experimental group: (1) Composition: Astragalus, Atractylodes macrocephala, Eleutherodactyla chinensis, Scutellaria barbata, Curcuma zedoaria, Trillium, Mother of Pearl, Fructus Aurantii Immaturus, made into a formula granule according to the composition of Chinese herbal medicine slices, 5 g / pack, 1 pack each time, 3 times a day, orally; (2) Folic acid tablet simulant, 5 mg / tablet, 1 tablet each time, 3 times a day, orally.

[0192] Control group: (1) Folic acid tablets, 5 mg / tablet, 1 tablet each time, 3 times a day, orally; (2) Formula granule simulation agent, 5 g / packet, 1 pack each time, 3 times a day, orally. The treatment course for both groups was 24 weeks.

[0193] The formula granules, folic acid tablets, and their simulation agents were provided by Guangdong Yifang Pharmaceutical Co., Ltd., and drug quality control was carried out.

[0194] 8. Sample size estimation

[0195] According to the preliminary test results, the effective rates of the control group and the experimental group were 65% and 35% respectively. The power 1-β = 0.80, the test level α = 0.05 (two-sided), the experimental group: control group = 1:1, using the following formula:

[0196]

[0197] The sample size was estimated using "Tests for Two Proportions" under the Proportions menu of the PASS2021 software. According to the pre-set parameters, after calculation using the PASS2021 software, it was found that 40 samples were required for each group. Taking into account the dropout rate (20%), a total of 100 subjects were included in this study. There were 3 clinical sub-centers in this study, and the random coding was generated by the stratified block randomization method. The First Affiliated Hospital of Guangzhou University of Chinese Medicine included 50 subjects, with 25 subjects in each experimental group and control group; Hainan Provincial Hospital of Traditional Chinese Medicine included 25 subjects, 12 in the experimental group and 13 in the control group; Zhongshan Hospital of Traditional Chinese Medicine included 25 subjects, 13 in the experimental group and 12 in the control group.

[0198] 9. Efficacy evaluation indicators

[0199] 9.1 Primary outcome measures

[0200] 9.1.1 Changes and reversal rates of OLGA and OLGIM stages before and after treatment:

[0201] The OLGA and OLGIM New Sydney staging standards were used to stage atrophy and IM, respectively, and divided into stages 0 to IV. See Table 12 for details. In order to evaluate the changes in the subjects' OLGA and OLGIM scores before and after treatment and their treatment effects, the reversal rate was used as an evaluation indicator. A decrease of ≥1 stage in the OLGA or OLGIM stage before and after treatment was defined as "reversal", while an increase of ≥1 stage was defined as "progression". The calculation formula for the reversal rate is: reversal rate (%) = (number of reversal cases after treatment / total number of cases) × 100%.

[0202] Table 12 OLGA and OLGIM New Sydney staging standards and scores

[0203]

[0204]

[0205] 9.2 Secondary Outcome Measures

[0206] 9.2.1 Spleen and Stomach Diseases-Chronic Gastritis Patient-Reported Outcome Scale (SSDPRO-CG) score:

[0207] ① Item score: Each item adopts a five-level scoring method. When scoring, positive items are directly scored from 1 to 5 points, and negative items are scored in reverse, that is, those who fill in the first level are scored 5 points, those who fill in the second level are scored 4 points, and so on. ② Calculation of domain, small aspect and total scale scores: Add up the scores of the items included in each domain and small aspect to get the score of the domain and small aspect. If there is a default value on the answer to the item, it is replaced by the average value. The sum of the scores of each domain is the total score of SSDPRO-CG.

[0208] 9.2.2 Safety indicators: routine blood test, routine urine test, routine stool test + occult blood, liver and kidney function test, and electrocardiogram.

[0209] 9.3 Evaluation time

[0210] Patients underwent visits at 0, 8, 16, and 24 weeks of treatment, for a total of 4 visits. A gastroscopy review was performed at the 24th week (the 4th visit).

[0211] 10. Statistical methods

[0212] (1) Descriptive statistics: SPSS 26.0 software was used for statistical analysis. Count data were expressed as frequency and composition ratio; measurement data that conformed to normal distribution were expressed as mean and standard deviation, and measurement data that did not conform to normal distribution were expressed as median or mode. (2) Difference test: SPSS 26.0 software was used for statistical analysis, and chi-square test was used for comparison between groups of count data. When the measurement data were normally distributed and the variance was equal, analysis of variance / analysis of variance with repeated measures was used; when the measurement data were not normally distributed or the variance was unequal, the rank sum test was used.

[0213] result:

[0214] 1. General

[0215] A total of 100 CAG patients were recruited in this clinical trial, with 50 in the experimental group and 50 in the control group. During the study, 18 subjects dropped out due to factors such as voluntary withdrawal and poor compliance (8 in the experimental group and 10 in the control group), and 82 subjects were finally included in the analysis (42 in the experimental group and 40 in the control group). There was no statistically significant difference in age, gender, smoking history, drinking history, family history of gastrointestinal tumors, and BMI between the two groups; see Table 13.

[0216] Table 13 Comparison of baseline data between the two groups of subjects

[0217]

[0218] 2. Comparison of efficacy of the main outcome measures

[0219] OLGA and OLGIM staging: There was no significant difference in OLGA and OLGIM staging between the two groups before treatment (P>0.05); there was a significant difference in OLGA staging between the two groups after treatment (P<0.05); there was no significant difference in OLGIM staging between the two groups after treatment (P>0.05); see Tables 14 and 15.

[0220] OLGA reversal rate: the control group was 32.50% (13 / 40), the experimental group was 64.29% (27 / 42), the difference between the two groups was statistically significant (x2 =8.284, P=0.004); OLGIM reversal rate: 25.00% (10 / 40) in the control group and 47.62% (27 / 42) in the experimental group. The difference in OLGIM reversal rate between the two groups was statistically significant (x 2 =4.518, P=0.033); see Table 16.

[0221] Table 14 Comparison of OLGA stages before and after treatment between the two groups of patients [cases (%)]

[0222]

[0223] Table 15 Comparison of OLGIM stages of the two groups of patients before and after treatment [cases (%)]

[0224]

[0225] Table 16 Comparison of reversal rates of OLGA and OLGIM between the two groups of patients [cases (%)]

[0226]

[0227] 3. Comparison of efficacy of secondary outcome indicators

[0228] As shown in Table 17, there were no significant differences in the physiological domain, independence, psychological domain, social environment domain, and total score between the two groups before treatment (P<0.05). After treatment, the physiological domain scores and total scores of both groups were significantly higher than those before treatment, and the difference was statistically significant (P<0.05); in addition, the experimental group had significantly higher scores in independence and psychological aspects, and the difference was statistically significant (P<0.05), while the control group did not observe this effect. No significant effect was observed in improving the social environment domain in both groups.

[0229] Table 17 Comparison of SSDPRO-CG scores between the two groups of patients before and after treatment (x±s, points)

[0230]

[0231] 4. Safety Observation Results

[0232] Before and after treatment, various safety indicators (blood routine, liver and kidney function, coagulation function, etc.) of the two groups of patients remained stable, with no obvious abnormalities; electrocardiogram results also showed no clinically significant changes.

[0233] The results showed that the reversal rates of OLGA and OLGIM in the experimental group were 64.29% and 47.62%, respectively, which were significantly higher than those in the control group (P<0.05). It not only effectively improved the pathological state of CAG patients, but also significantly improved the quality of life scores of patients in the physiological (clinical manifestations), independence and psychological fields. Especially in terms of mental health, patients showed more positive emotional states, significantly alleviated anxiety symptoms and better psychological adaptability. The above results suggest that the formula granules also have good therapeutic effects in improving patients' clinical symptoms and mental state.

[0234] Among them, the effective extract of Astragalus membranaceus can inhibit the proliferation and migration of gastric cancer cells, promote their apoptosis, inhibit angiogenesis, promote the repair of gastric mucosal damage induced by MNNG in rats, and prevent and treat gastric mucosal atrophy, intestinal metaplasia and intraepithelial neoplasia; Astragaloside IV, the main active substance of Astragalus membranaceus, reduces the expression of CXCL12 by inhibiting the JAK2 / STAT3 signaling pathway, inhibits the infiltration of inflammatory cells, and improves the inflammation of gastric mucosa in gastritis mice. Atractylodes macrocephala has the effect of enhancing the proliferation and repair of gastric mucosal cells and promoting the secretion of gastric mucosal cells. Pharmacological studies have found that five-fingered peach has the effects of repairing gastric mucosal damage, improving immune function, anti-oxidation, bactericidal and anti-inflammatory.

[0235] Studies have shown that Curcuma and Scutellaria barbata can inhibit tumor cell proliferation or apoptosis, inhibit intestinal metaplasia and intraepithelial neoplasia, and thus reduce the risk of atrophic cancer. Trillium has anti-inflammatory, analgesic, and anti-tumor pharmacological effects; Citrus aurantium contains flavonoids, alkaloids, volatile oils, coumarins and other ingredients, which have multiple pharmacological effects such as promoting gastric motility, anti-anxiety, anti-inflammatory, antibacterial, and antioxidant. Mother of pearl, its ingredients have multiple effects such as anticonvulsant and sedative hypnosis, improving memory impairment, anti-inflammatory and anti-apoptotic, and enhancing immunity.

[0236] As described above, the basic principle, main features and advantages of the present invention are well described. The above embodiments and descriptions are only descriptions of the preferred embodiments of the present invention, and the present invention is not limited by the above embodiments. Without departing from the spirit and scope of the present invention, various changes and improvements made by ordinary technicians in this field to the technical solution of the present invention should fall within the protection scope determined by the present invention.

Claims

1. A Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia, characterized in that: The formula is: Astragalus, quinquefolius, Atractylodes macrocephala, Scutellaria barbata, Curcuma zedoaria, Trillium, mother of pearl, Citrus aurantium.

2. A Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia according to claim 1, characterized in that: By weight, the formula is: 15g of Astragalus, 20g of Prunus quinquefolia, 10g of Atractylodes macrocephala, 20g of Scutellaria barbata, 10g of Curcuma, 10g of Trillium, 15g of Mother of Pearl, and 10g of Citrus aurantium.

3. The Chinese medicine composition for treating chronic atrophic gastritis with intestinal metaplasia according to any one of claims 1 to 2, characterized in that: Used for preparing Chinese medicine preparations or Chinese medicine pieces or Chinese medicine freeze-dried powder for treating chronic atrophic gastritis with intestinal metaplasia.

4. A method for preparing the lyophilized powder of traditional Chinese medicine for treating chronic atrophic gastritis with intestinal metaplasia according to claim 3, characterized in that: include: Step 1: Weigh 10-30 parts of Astragalus, 15-30 parts of Prunus mume, 10-30 parts of Atractylodes macrocephala, 10-30 parts of Scutellaria barbata, 5-20 parts of Curcuma, 5-20 parts of Trigonella ternata, 5-30 parts of Mother of Pearl and 5-20 parts of Fructus Aurantii Immaturus respectively; Step 2: Mix and crush the astragalus, quinquefolius, atractylodes, scutellaria barbata, zedoaria, trillium, mother-of-pearl and fructus aurantii weighed in step 1 to obtain crushed drugs, and then sieve to obtain coarse drug powder; Step 3: Soak the crude drug powder obtained in step 2 in water for 1 to 2 hours, boil it over high heat, and then decoct it over low heat for 1 to 2 hours, filter and separate it to obtain a filter residue and a primary filtrate; Step 4: Add water to the filter residue obtained in step 3, boil for 1 to 2 hours, filter and separate to obtain a secondary filtrate, and combine the secondary filtrate with the primary filtrate to obtain a water extract; Step 5: The water extract obtained in step 4 is concentrated under reduced pressure at 60-65° C. to obtain concentrated medicine, and then freeze-dried in a vacuum to obtain lyophilized powder of traditional Chinese medicine.

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