Use of notopterygium root and fish in preparing a medicine for preventing and / or treating precancerous lesions of gastric cancer

Abstract

The application discloses a use of Notopterygium root fish in preparation of a medicine for preventing and / or treating precancerous lesions of gastric cancer. The animal experiment research proves that the Notopterygium root fish and a traditional Chinese medicine prescription containing the Notopterygium root fish, i.e. Notopterygium root pill, can improve the intestinal epithelial metaplasia and dysplasia of gastric mucosa of GPL rats, promote the apoptosis of gastric mucosa epithelial lesion cells, protect the gastric mucosa and restore the function of the gastric mucosa. The Notopterygium root fish or the Notopterygium root pill has different degrees of curative effects on the precancerous lesions of gastric cancer such as gastric mucosa intestinal metaplasia and dysplasia, and has practical popularization and application values.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and particularly relates to use of Notopterygium in preparing medicines for preventing and / or treating gastric precancerous lesions. Background Art

[0002] The multi-step Correa cascade of gastric cancer states that, in most cases, gastric cancer develops through a progression from normal mucosa to chronic gastritis, mucosal atrophy, intestinal metaplasia, dysplasia, and finally cancer. According to this theory, gastric precursor lesions (GPLs) are digestive system diseases that precede the development of gastric cancer. These primarily include dysplasia and intestinal metaplasia, histopathological changes in the gastric mucosa that are prone to cancerous development. Early intervention in GPLs has a significant potential to reverse malignant transformation of the gastric epithelium and prevent the development of gastric cancer.

[0003] Qianghuo fish is the entire stream salamander (Batrachuperus pinchonii (David)) of the family Microhylidae. It has a pungent and salty taste and a neutral nature. It enters the liver and stomach meridians, has the effect of promoting qi and relieving pain, and is commonly used clinically to treat stomachache, joint pain, traumatic injuries, and other conditions. Currently, there are traditional Chinese medicine prescriptions containing Zhishi (Fructus Aurantii Immaturus), Atractylodes Macrocephala, and Qianghuo fish for treating stomachache, bloating, nausea, etc., such as Weixiao Mixture, which is composed of 13 traditional Chinese medicines including Zhishi (Fructus Aurantii Immaturus), Atractylodes Macrocephala, Qianghuo fish, Coptis chinensis, Evodia rutaecarpa, and Corydalis chinensis. However, there are no reports of studies using Qianghuo fish as the sole active ingredient, or Qianghuo fish combined with Atractylodes Macrocephala as the sole active ingredient, for the treatment of gastric precancerous lesions. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides the use of Notopterygium wilfordii as the sole active ingredient in the preparation of a medicament for preventing and / or treating gastric precancerous lesions.

[0005] The present invention also provides use of Notopterygium wilfordii combined with Atractylodes macrocephala as the sole active ingredient in preparing a medicine for preventing and / or treating gastric precancerous lesions.

[0006] Furthermore, the drug is a drug for preventing and / or treating gastric mucosal atrophy, gastric mucosal intestinal metaplasia and / or gastric mucosal dysplasia.

[0007] Furthermore, the mass ratio of the Notopterygium wilfordii to Atractylodes macrocephala is 2-8:4-10.

[0008] Furthermore, the mass ratio of the Notopterygium wilfordii to Atractylodes macrocephala is 3:6.

[0009] Furthermore, the Atractylodes macrocephala is fried Atractylodes macrocephala with bran.

[0010] The present invention also provides a medicine for preventing and / or treating gastric precancerous lesions, which is a preparation prepared by taking Notopterygium incisum or a traditional Chinese medicine composition containing Notopterygium incisum as an active ingredient and adding pharmaceutically acceptable excipients.

[0011] Furthermore, the preparation is an oral preparation; the oral preparation is a solution, granules, pills, capsules or granules.

[0012] Furthermore, the Chinese medicine composition is prepared from the following raw materials in parts by weight:

[0013] 4-10 portions of stir-fried Atractylodes macrocephala with bran and 2-8 portions of Notopterygium wilfordii.

[0014] Furthermore, the Chinese medicine composition is prepared from the following raw materials in parts by weight:

[0015] 6 portions of stir-fried Atractylodes macrocephala with bran and 3 portions of Notopterygium wilfordii.

[0016] Furthermore, the drug is a drug for preventing and / or treating gastric mucosal atrophy, gastric mucosal intestinal metaplasia and / or gastric mucosal dysplasia.

[0017] The present invention discloses the use of Qianghuo fish in the preparation of a medicament for preventing and / or treating gastric precancerous lesions. Animal experiments have demonstrated that Qianghuo fish and Qiangshu Pills, a traditional Chinese medicine formula containing the fish, can improve intestinal metaplasia and dysplasia in the gastric mucosa of GPL rats, promote apoptosis of cells in gastric mucosal epithelial lesions, protect the gastric mucosa, and restore gastric mucosal function. The present invention demonstrates varying degrees of efficacy in treating gastric precancerous lesions such as intestinal metaplasia and dysplasia in the gastric mucosa, demonstrating practical application value.

[0018] Obviously, based on the above contents of the present invention, according to common technical knowledge and customary means in this field, without departing from the above basic technical ideas of the present invention, other various forms of modifications, replacements or changes can be made.

[0019] The following further describes the above content of the present invention in detail through specific embodiments in the form of examples. However, this should not be construed as limiting the scope of the above subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention fall within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 :HE staining of gastric mucosal tissue of GPL animals

[0021] Figure 2 : AB-PAS staining of gastric mucosal tissue of GPL animals

[0022] Figure 3 :TUNEL expression in gastric mucosal epithelial cells of GPL animals

[0023] Figure 4 :Expression of HIF-α protein in gastric mucosal epithelial cells of GPL animals

[0024] Figure 5 :VEGF protein expression in gastric mucosal epithelial cells of GPL animals DETAILED DESCRIPTION

[0025] Example 1 Study on the therapeutic effects of Qianghuo fish and Qianghuo fish modified prescription on gastric precancerous lesions

[0026] 1. Experimental drugs

[0027] Qianghuo Fish: Ingredients: 12g Qianghuo Fish. Place the Qianghuo Fish medicinal material in a suitable container. Soak the medicinal material in 10 times the amount of purified water for 30 minutes. Decoction the material in three batches: boil over high heat for the first decoction, then simmer over low heat for 20 minutes. The second and third decoctions last for 25 minutes each. Filter and concentrate the three decoctions. Add excipients to prepare a Qianghuo Fish preparation containing 1g of the crude drug per 1g. Store in a refrigerator at 4°C until ready for use.

[0028] Qiangshu Pills: Formula: 6g stir-fried Atractylodes macrocephala with bran and 3g Notopterygium incisum. Preparation: Weigh the raw materials according to the formula. Soak the stir-fried Atractylodes macrocephala with bran in 10 times the amount of purified water for 30 minutes. Decoction is divided into three steps: first, boil over high heat, then simmer over low heat for 20 minutes. Second and third decoctions are 25 minutes each. Filter the three decoctions and concentrate to 1g·mL. -1 , grind the Notopterygium chinense fish into powder and mix it, add auxiliary materials to obtain Qiangshu pills containing 1g of raw medicine per 1g, and store it in a refrigerator at 4℃ for later use.

[0029] Gastric Atrophy Elimination: Recipe: Adenophora australis 12g, processed Citrus aurantium 18g, Poria 15g, Curcuma 8g, Atractylodes macrocephala 12g, and Notopterygium incisum 5g. Soak the Chinese medicinal materials in 10 times the amount of pure water for 30 minutes, and decoct them in 3 times. Boil the first decoction over high heat and then simmer for 20 minutes over low heat. The second and third decoctions are 25 minutes each. Filter and concentrate the three medicinal liquids, add auxiliary materials to make gastric atrophy elimination solution containing 3g of raw medicinal materials per 1g, and store in a refrigerator at 4℃ for later use.

[0030] Folic acid: Produced by Changzhou Pharmaceutical Factory Co., Ltd., Approval Number: National Medicine Standard H32023302

[0031] 2. Experimental Materials

[0032] 2.1 Experimental Reagents

[0033] Rat PGI Elisa Kit, Rat GAS17 Elisa Kit, and Rat PGE2 Elisa Kit were purchased from mlbio with catalog numbers ml059188, ml059375, and ml003036, respectively; VEGF Receptor 3 Rabbit pAb (GB112339, Servicebio); and HIF-1α Rabbit (YT788, Guidechem).

[0034] 2.2 Experimental animals

[0035] SPF-grade SD rats, half male and half female, aged 7 to 8 weeks, weighing (190 ± 10) g, were selected. The rats were purchased from Chengdu Dashuo Experimental Animal Co., Ltd. with certificate number SCXK (Sichuan) 2020-030.

[0036] 3. Experimental Methods

[0037] 3.1 Experimental groups and drug administration

[0038] The rats were divided into a blank group (n=10) and a model group (n=52). The blank group was fed with conventional diet. The model group was treated with MNNG-ammonia compound modeling method to establish the GPL rat model: 0.1% ammonia water was given and drinking water was available. -1 The rats were gavaged with MNNG solution twice a week, fasting for 24 hours each time. At the end of the eighth week, two animals were randomly selected from the model group. The gastric mucosa at the corpus-antrum junction was obtained and stained with hematoxylin and eosin (H&E) to confirm early GPL lesions under a light microscope. The model group rats were then randomly divided into five groups (n=10): model group, folic acid group, Weiweixiao group, Qiangshu pill group, and Qianghuoyu group. From the ninth to the 20th week, the folic acid group received 1.8 mg / kg / day; the Weiweixiao group received 6.3 g / kg / day; the Qiangshu pill group received 0.81 g / kg / day; and the Qianghuoyu group received 1.08 g / kg / day. Rats in the blank and model groups received 10 ml / kg of normal saline daily for 12 weeks. At the end of week 20, all rats were humanely euthanized with sodium pentobarbital (140 mg / kg) after fasting for 12 h.

[0039] Table 1 Dosage table for each group

[0040]

[0041] 3.2 HE staining of gastric mucosal tissue

[0042] After a 12-hour fast, rats were anesthetized with sodium pentobarbital (140 mg / kg intraperitoneally). Gastric tissue was removed and fixed overnight in 10% neutralized formalin. The tissue was then dehydrated with alcohol and xylene. 3-μm paraffin-embedded sections were prepared and stained with hematoxylin and eosin (H&E) according to standard procedures. Histopathological changes in the gastric mucosal epithelium were observed under a light microscope (IX71; Olympus Corporation).

[0043] 3.3 AB-PAS staining of gastric mucosal tissue

[0044] After a 12-hour fast, rats were anesthetized with sodium pentobarbital (140 mg / kg intraperitoneally). Gastric tissue was removed and fixed overnight in 10% neutralized formalin. The tissue was then dehydrated with alcohol and xylene. 3 μm paraffin-embedded sections were then prepared and the type of intestinal metaplasia was determined using Alcian blue and periodic acid-Schiff staining (AB-PAS). Neutral mucin in normal mucosa stains magenta, while acidic mucin in intestinal lesions stains blue. The extent of lesions was observed under a light microscope (IX71; Olympus).

[0045] 3.4 Gastric mucosal PGI, GAS17, and PGE2 detection

[0046] After washing and homogenizing the gastric mucosal tissue, centrifuge at 5000 rpm for 5 minutes and collect the supernatant. Set up standard and sample wells. Add 100 μL of horseradish peroxidase-labeled detection antibody to each well, except for the blank well. Seal the wells with sealing film and incubate at 37°C in a water bath or incubator for 60 minutes. Wash five times, then add 50 μL each of substrates A and B to each well and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm.

[0047] 3.5 Immunohistochemical detection of HIF-α and VEGF protein expression

[0048] Paraffin-embedded gastric tissue was cut into 3-μm-thick sections, dewaxed with xylene at room temperature, and then rehydrated with an ethanol series. Sections were heated at 97°C for 20 minutes. After peroxidase blocking with 3% hydrogen peroxide for 15 minutes and 3% BSA for 30 minutes, sections were incubated with primary antibodies against HIF-α and VEGF proteins overnight at 4°C. Sections were then exposed to HRP / Fab polymer conjugates at room temperature for 30 minutes, followed by staining with 3,3'-diaminobenzidine solution for 5 minutes and counterstained with hematoxylin for 20 seconds at room temperature. Sections were dehydrated and mounted. Microscopic examination and image acquisition and analysis were performed. Three random fields of view were selected and photographed from each section. Expression levels were quantified by integrated optical density (IOD) using Image Pro Plus 6.0 software (Media Cybernetics, Inc.).

[0049] 3.6 Statistical analysis

[0050] All experimental data were expressed as mean ± standard deviation (mean ± SD). One-way analysis of variance was used for comparison between data groups, and one-way analysis of variance was used for comparison between multiple groups. Turkey method was used when variances were equal, and Dunnett T3 test was used when variances were unequal. P < 0.05 indicated statistical significance.

[0051] 4. Experimental Results

[0052] 4.1 Qianghuoyu and Qiangshu pills improve gastric mucosal atrophy and dysplasia in GPL rats

[0053] Figure 1 As shown in Figure 2, after HE staining, light microscopy revealed normal gastric epithelial glands and structures, with virtually no inflammatory infiltration. In the model group, GPL rats showed atrophy and reduction of gastric epithelial glands, asymmetric basement membrane thickness, and disordered gastric epithelial gland structure, with enlarged, darkly stained, and crowded nuclei. In the treatment groups, gastric mucosal atrophy and dysplasia were alleviated to varying degrees. As shown in Table 2, the SL score was used to assess the extent of gastric mucosal lesions in rats. Compared with the blank group, the extent of gastric mucosal dysplasia in the model group GPL rats was significantly improved (P < 0.01), indicating successful modeling. Compared with the model group, the extent of gastric epithelial atrophy and dysplasia in the folic acid group was slightly improved, but the difference was not statistically significant (P > 0.05). Significant improvements in gastric epithelial atrophy and dysplasia were observed in the Weiweixiao, Qiangshuwan, and Qianghuoyu groups (P < 0.01 or P < 0.05). Compared with the Qianghuo fish group, the improvement of gastric epithelial atrophy and dysplasia in the Weiweixiao group and Qiangshu pill group was more significant, with statistical difference (P<0.05). In summary, Qiangshu pill and Qianghuo fish can improve the atrophy and dysplasia of the gastric mucosal epithelium in GPL rats, and the efficacy of Qiangshu pill is relatively more significant. See Table 2 and Figure 1 .

[0054] Table 2 Analysis of the degree of gastric mucosal atrophy and dysplasia

[0055]

[0056] Note: Compared with the blank group, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; compared with the group of Notopterygium spp. ▲ P<0.05, ▲▲ P<0.01

[0057] 4.2 Qianghuoyu and Qiangshu pills improve gastric mucosal intestinal metaplasia in GPL rats

[0058] AB-PAS staining was used to detect intestinal metaplasia of gastric mucosa. Figure 1As shown, no blue-purple positive cells were observed in the gastric mucosa of rats in the blank group. Compared with the blank group, numerous blue or purple plaques were observed in the gastric mucosa of rats in the model group (GPL), indicating significant intestinal metaplasia. Compared with the model group, the folic acid group showed a slight decrease in intestinal metaplasia. The blue-purple plaques in the gastric mucosa of rats in the Weiweixiao, Qiangshuyu, and Qiangshuwan groups were significantly reduced, indicating a reduction in intestinal metaplasia. As shown in Table 3, the SL score was used to assess the degree of intestinal metaplasia in rats. Compared with the blank group, the model group showed significant intestinal metaplasia, with a statistically significant difference (P < 0.01), indicating successful modeling. Compared with the model group, the intestinal metaplasia in the gastric mucosa of rats in the Weiweixiao, Qiangshuyu, and Qiangshuwan groups was significantly improved (P < 0.01). The degree of intestinal metaplasia in the gastric mucosa of rats in the folic acid group was slightly improved, but not statistically significant (P > 0.05). Compared with the Qianghuo fish group, the improvement of gastric mucosal intestinal metaplasia in the Weiweixiao group and Qiangshu pill group was more significant, with statistical difference (P<0.05). In summary, the Qiangshu pill group and the Qianghuo fish group both had a significant effect on improving gastric mucosal intestinal metaplasia in GPL rats, among which the efficacy of Qiangshu pill was relatively more significant. See Table 3 and Figure 2 .

[0059] Table 3 Analysis of the degree of intestinal metaplasia in gastric mucosa

[0060]

[0061] Note: Compared with the blank group, ** P<0.01; compared with the model group, # P<0.05, ## P<0.01; compared with the group of Notopterygium spp. ▲ P<0.05, ▲▲ P<0.01

[0062] 4.3 Qianghuoyu and Qiangshu pills improve the levels of PGI, GAS17, and PGE2 in the gastric mucosal epithelium of GPL rats

[0063] To further verify the effects on gastric mucosal function, ELISA was used to detect gastric mucosal PGI, GAS17, and PGE2 levels. As shown in Table 4, compared with the blank group, the levels of PGI and PGE2 in the gastric mucosal epithelial tissue of the GPL rats in the model group were significantly decreased (P < 0.01), while the level of GAS17 was significantly increased (P < 0.01). Compared with the model group, the levels of PGI and PGE2 in the gastric mucosal epithelial tissue of the rats in the Weiweixiao, Qianghuoyu and Zhizhu groups, and Qianghuoyu groups were significantly increased (P < 0.01), while the level of GAS17 was decreased (P < 0.01). Compared with the Qianghuoyu group, the levels of PGI in the gastric mucosal epithelial tissue of the rats in the Weiweixiao and Qiangshuwan groups were more significantly increased, with statistically significant differences (P < 0.01). The above results suggest that the function of gastric mucosa in GPL declines, which promotes the proliferation and deterioration of gastric precancerous lesion cells. However, Qiangshu Pills and Qianghuo fish can regulate the levels of PGI, GAS17 and PGE2 in the gastric mucosa of GPL, protect the function of gastric mucosa and promote the recovery of gastric mucosa.

[0064] Table 4 PGI, GAS17, and PGE2 levels in gastric mucosal epithelial tissue

[0065]

[0066] Note: Compared with the blank group, ** P<0.01, compared with the model group, # P<0.05, ## P<0.01; compared with the group of Notopterygium spp. ▲ P<0.05, ▲▲ P<0.01

[0067] 4.4 Qianghuoyu and Qiangshu pills induce apoptosis of gastric mucosal epithelial cells in GPL rats

[0068] In order to further detect the number of apoptotic cells in rat gastric mucosal tissue, we used TUNEL staining. Figure 3 As shown in Table 5, compared with the blank group, the apoptosis rate of gastric epithelial cells in the model group was significantly decreased (P<0.01), while the apoptosis rate of gastric epithelial cells in the treatment groups (weiweixiao group, Qiangshu pill group, and Qianghuoyu group) was significantly increased compared with the model group (P<0.01). The results suggest that Qianghuoyu and Qiangshu pill can induce apoptosis of gastric epithelial cells in GPL rats.

[0069] Table 5 TUNEL staining results of gastric mucosal epithelial cells

[0070]

[0071] Note: Compared with the blank group, ** P<0.01, compared with the model group, # P<0.05,## P<0.01

[0072] 5.5 Qianghuoyu and Qiangshu pills reduce the levels of HIF-α and VEGF proteins in the gastric mucosal epithelium of GPL rats

[0073] HIF-α and VEGF are closely related to the occurrence and development of tumors. High expression of both can promote the growth, invasion and metastasis of gastric cancer. Figure 4-5 Table 6 shows that compared with the blank group, diffuse, strong cytoplasmic expression of HIF-α and VEGF proteins was found in the gastric mucosal epithelium of rats in the model group (P < 0.01). Compared with the model group, the positive expression of HIF-α and VEGF proteins in the gastric mucosal epithelium of rats in the folic acid group was reduced, with statistically significant differences (P < 0.01 or P < 0.05). The protein expression of HIF-α and VEGF in the gastric mucosal epithelium of rats in the Weiweixiao group, Qiangshuwan group, and Qianghuoyu group was significantly reduced, with statistically significant differences (P < 0.01). Compared with the Qianghuoyu group, the protein expression of HIF-α and VEGF in the gastric mucosal epithelium of rats in the Weiweixiao group and Qiangshu group was more significantly reduced, with statistically significant differences (P < 0.01 or P < 0.05). The above results show that Qiangshu Pills and Qianghuo Fish can significantly reduce the expression of HIF-α and VEGF proteins in the gastric mucosal epithelium of GPL rats and prevent the further development of GPL, among which the effect of Qiangshu Pills is relatively more significant.

[0074] Table 6 Expression of HIF-α and VEGF proteins in gastric mucosal tissue

[0075]

[0076] Note: Compared with the blank group, **P<0.01; compared with the model group, #P<0.05, ##P<0.01; compared with the Qianghuo fish group, ▲P<0.05, ▲▲P<0.01

[0077] In summary, animal studies have demonstrated that Qianghuo fish and its traditional Chinese medicine formula, Qiangshu Pills, can improve intestinal metaplasia and dysplasia in the gastric mucosa of GPL rats, promote apoptosis of gastric epithelial cells, protect the gastric mucosa, and restore gastric mucosal function. The present invention demonstrates that using Qianghuo fish or Qiangshu Pills for treating gastric precancerous lesions such as intestinal metaplasia and dysplasia has varying degrees of efficacy and possesses practical application value.

Claims

1. Use of Qianghuo fish combined with bran-fried Atractylodes macrocephala as the sole active ingredient in the preparation of a drug for treating gastric precancerous lesions; The mass ratio of the Qianghuo fish to the bran-fried Atractylodes macrocephala is 3:6; The medicine is a medicine for treating gastric mucosal atrophy, gastric mucosal intestinal metaplasia and / or gastric mucosal dysplasia.

2. A drug for treating gastric precancerous lesions, characterized by: The preparation is prepared by using qianghuo fish or qianghuo fish combined with bran-fried atractylodes as the only active ingredient and adding pharmaceutically acceptable excipients; the preparation is an oral preparation; the oral preparation is a solution, granule, pill or capsule The mass ratio of the Qianghuo fish to the bran-fried Atractylodes macrocephala is 3:6; The medicine is a medicine for treating gastric mucosal atrophy, gastric mucosal intestinal metaplasia and / or gastric mucosal dysplasia.

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