CN111714523A - Application of a kind of Dunaliella in the prevention or treatment of colon cancer - Google Patents

Abstract

The invention relates to the application of Dunaliella in the prevention or treatment of colon cancer, and belongs to the technical field of biomedicine and microorganisms. The Dubosiella newyorkensis NYU-BL-A4 used in the present invention can reduce the expression of verification factors related to colon cancer at the molecular level, thereby inhibiting the proliferation of colon cancer cells and reducing the viscera of colon cancer patients Weight and number of colonic adenomas. The performance of this strain of inhibiting or reducing the expression level or proliferation ability of colon cancer-related factors, coupled with its prebiotic ability, can be used to prepare drugs that alleviate the occurrence of intestinal inflammatory factors and inhibit the proliferation of related cells. It is beneficial to regulate intestinal immunity and maintain intestinal health, and has broad market prospects.

Description

Application of a kind of Dunaliella in the prevention or treatment of colon cancer

technical field

The invention relates to the application of Dunaliella in the prevention or treatment of colon cancer, and belongs to the technical field of biomedicine and microorganisms.

Background technique

Colorectal cancer is a malignant tumor that occurs in the colon or rectum of the lower gastrointestinal tract of the human body. According to the statistics of the International Center for Research on Cancer of the World Health Organization in 2017, colorectal cancer ranks third in the incidence of all malignant tumors in the world, and the mortality rate is the third. It ranks fourth, and the incidence and mortality are on the rise. The incidence of colorectal cancer in China ranks third in the incidence of malignant tumors. Colorectal cancer has developed into one of the serious diseases that threaten human health. Colorectal cancer is a multifactorial disease that involves many links in the process of occurrence and development. Its causative factors include genetic and environmental factors, and living and eating habits are also highly correlated with colorectal cancer. The symptoms of colorectal cancer are not obvious in the early stage, but as the tumor enlarges, patients will experience changes in bowel habits, diarrhea, alternating blood in the stool with local abdominal pain, and constipation. In the advanced stage, symptoms such as anemia and weight loss occur. Studies have shown that "high-fat, high-protein, high-calorie, low-fiber" diet and smoking and drinking habits will greatly increase the risk of disease.

In the human gut, especially the colon, there is a complex micro-ecosystem composed of a large number of microorganisms, which is called gut microbiota. The number of bacteria accounts for the vast majority. The normal human gut contains different types and quantities of bacteria, including beneficial bacteria, harmful bacteria and neutral bacteria. The intestinal flora participates in many basic physiological activities of the human body, such as promoting the development and maturation of the intestinal tract, promoting the metabolism and absorption of substances, synthesizing vitamins, stimulating immune function, resisting the invasion of pathogens, maintaining the intestinal barrier, and degrading cholesterol. Intestinal flora can achieve anti-tumor effects by degrading and removing carcinogenic factors in the body and activating anti-tumor cytokines in the body.

Based on a large-scale population-based group comparison analysis, it was found that dysbiosis of gut microbiota is prevalent in patients with colorectal cancer. During the occurrence and development of colorectal cancer, the structure of intestinal flora changes. There are great differences in the composition of gut microbiota between people in high-incidence areas and low-incidence areas of colorectal cancer, indicating that changes in gut microbiota are closely related to the occurrence and development of colorectal cancer. Traditional surgical treatment combined with chemotherapy and radiation therapy has serious toxic and side effects on patients. Although the five-year survival rate is improved, it seriously affects the quality of life of patients. Probiotics in the intestinal flora have stable flora abundance. , The effect of inhibiting inflammatory diseases. Regulating intestinal flora is one of the important means to prevent and treat colorectal cancer. Using these probiotics to maintain intestinal homeostasis can reduce the occurrence of intestinal inflammatory diseases. As an important part of the intestinal flora, probiotics in the intestinal tract can form intestinal mucosa in the intestinal tract and prevent the adhesion and invasion of pathogenic bacteria. Foods containing Lactobacillus and Bifidobacterium can be used as prebiotics to repair intestinal epithelial mucosa, inhibit the proliferation of tumor cells, and have certain preventive and therapeutic effects on colorectal tumors. It can be seen that probiotics play a very important role in the human body's anti-cancer and anti-cancer, but currently there are few anti-cancer and anti-cancer drugs prepared from probiotics on the market.

Therefore, additional research and discovery of new functional bacterial genera are necessary to further support the clinical use of probiotics as a preventive treatment for colorectal tumors.

SUMMARY OF THE INVENTION

The occurrence of colorectal cancer (CRC) is affected by both genetic factors and environmental factors. Most patients with colorectal cancer have mutations in the tumor suppressor gene Apc gene. Therefore, those with Apc gene mutation can spontaneously form adenomatous polyps The Apc ^min/+ mouse is a good animal model to study the mechanism of bowel cancer. Bowel carcinogenesis is influenced by both genetic and environmental factors. Although 5-15% of bowel cancers are attributed to genetic factors, the vast majority of bowel cancers are sporadic and occur along the adenoma-adenocarcinoma pathway. The most important environmental factors affecting the occurrence of colorectal cancer include diet, microbial exposure and host immunity. Studies have found that high-fat diet mediates the imbalance of intestinal flora structure, and mice with intestinal cancer susceptibility genes significantly increase the intestinal tumor load after ingesting high-fat diets. Based on this, the present invention adopts the intestinal cancer susceptibility gene Apc. ^Min/+ mice were added with high-fat diet to establish a colon cancer mouse model to illustrate the effect of probiotics in the prevention or treatment of colon cancer.

As a new genus isolated and identified from biological samples, Dubosiella has shown effects such as regulating metabolism in the body, improving intestinal immunity and promoting the body's resistance to inflammatory diseases, and can affect a variety of life activities of individuals.

In order to solve the existing problems, the present invention provides the application of Dubosiella newyorkensis or a probiotic preparation containing Dubosiella in preparing a product for preventing, alleviating or improving colon cancer.

In one embodiment of the present invention, the Dunaliella is NYU-BL-A4, which is published in the patent with the publication number of US2018125900A1.

In one embodiment of the present invention, the probiotic preparation is used to improve the intestinal immunity of the body and inhibit the proliferation activity of intestinal cancer cells.

In one embodiment of the present invention, the product is a probiotic formulation that can be used as a dietary supplement and therapeutic agent.

In one embodiment of the present invention, in addition to Dunaliella, the probiotic preparation contains other excipients, including but not limited to excipients or food additives; the content of the Dunaliella in the probiotic preparation It is 1.0×10 ⁷ to 1.0×10 ¹⁰ cfu/mL or 1.0×10 ⁷ to 1.0×10 ¹⁰ cfu/g.

In one embodiment of the present invention,

The product includes a medicament or a pharmaceutical composition for use in at least one of (a) to (d):

(a) relieve spleen inflammation;

(b) reducing the number of colonic adenomas;

(c) inhibit colon cancer cell proliferation;

(d) Regulates the expression of inflammatory factors associated with colon cancer.

In an embodiment of the present invention, the content of the Dunaliella in the medicine or the pharmaceutical composition is 1.0×10 ⁷ to 1.0×10 ¹⁰ cfu/mL or 1.0×10 ⁷ to 1.0×10 ¹⁰ cfu/g.

In one embodiment of the present invention, the inflammatory factors are Tnf-α, Cox2, IL-6, and interleukin-1β.

In one embodiment of the present invention, the medicament or pharmaceutical composition further comprises a pharmaceutically acceptable excipient.

In one embodiment of the present invention, the pharmaceutically acceptable excipient refers to any diluent, adjuvant and/or carrier that can be used in the pharmaceutical field.

In one embodiment of the present invention, the product includes but is not limited to food, health product drink, enteral nutrition preparation, dietary supplement, veterinary medicine or feed additive.

In one embodiment of the present invention, the food, health product drink, enteral nutrition preparation, dietary supplement, veterinary medicine or feed additive further contains the technical field that those skilled in the art appropriately select ingredients according to the dosage form or purpose of use A common ingredient that can be used with other ingredients.

In one embodiment of the present invention, the conventional adjuvants include one or more of fillers, flavoring agents, binders, disintegrants, lubricants, antacids, and nutritional fortifiers.

Beneficial effects:

The Dubosiella newyorkensis NYU-BL-A4 in the present invention has a close relationship with colon cancer, and it can reduce the expression of the verification factors related to colon cancer at the molecular level, thereby inhibiting the proliferation of colon cancer cells, Decrease organ weight and number of colon adenomas in colon cancer patients. The performance of this strain of inhibiting or reducing the expression level or proliferation ability of colon cancer-related factors, coupled with its prebiotic ability, can be used to prepare drugs that alleviate the occurrence of intestinal inflammatory factors and inhibit the proliferation of related cells. It is beneficial to regulate intestinal immunity and maintain intestinal health, and has broad market prospects.

Description of drawings

Fig. 1 is a graph showing the effect of Dubosiella newyorkensis NYU-BL-A4 on the organ weight of colon cancer mice.

Figure 2 is a graph showing the effect of Dubosiella newyorkensis NYU-BL-A4 on the number of colonic adenomas.

Figure 3 is a graph showing the effect of Dubosiella newyorkensis NYU-BL-A4 on colon cancer cell proliferation.

Fig. 4 is a graph showing the effect of Dubosiella newyorkensis NYU-BL-A4 on the expression levels of inflammatory factors induced by colon cancer.

Detailed ways

Mouse C57BL/6J-ApcMin/Nju (Apc ^min/+ ) was purchased from Shanghai Slack Laboratory Animal Co., Ltd.

The high-fat feed was purchased from Nantong Trofee Biological Co., Ltd. (TP23300), which was a high-fat purified feed with a fat energy supply ratio of 60%.

Reverse transcription cDNA synthesis: use Takara's reverse transcription kit (Cat. No.: RR036A).

Example 1: Dubosiella newyorkensis NYU-BL-A4 reduces organ weight in mice with colon cancer

Dubosiella newyorkensis (Dubosiella newyorkensis) NYU-BL-A4 culture, gavage preparation: bacterial strains were spread on Brucella agar solid medium supplemented with 5mL/100mL defibrillated Mianyang blood, and cultured at 37°C in an anaerobic environment 72 hours; or in MTGE broth liquid medium at 37°C for 48 hours. Cultivation was stopped after entering log phase ( _OD600 = 0.5). After the medium suspension was centrifuged at 3000 rpm for 5 min, the lower viable bacterial pellet was mixed with anaerobic PBS in a strict anaerobic environment to prepare a bacterial solution with a concentration of 3.0×10 ⁹ cfu/mL for animal and cell experiments. Bacterial liquid is now prepared before the mouse to ensure the activity of the strain. The strains were stored in a -80°C long-term refrigerator.

The animal experiment method in this example was to select 30 4-week-old male Apc ^min/+ mice, weighing about 18-20 g. All mice were raised in the Experimental Animal Center of Jiangnan University in accordance with the standard operating procedures for raising SPF mice, and they were allowed to eat irradiated sterilized feed and water ad libitum. During the feeding period, the food and water intake, activity level, fur gloss and fecal characteristics of each mouse were paid attention to every day, and the mice were weighed and recorded every week.

After 7 days of acclimatization, 30 mice were randomly divided into 3 groups, 10 mice in each group, divided into:

(1) The Dubosiella intervention group (Dubosiella) consuming a high-fat diet, the mice were fed a high-fat diet and water ad libitum, and the mice received 0.2 mL of live Dubosiella solution 3.0×10 by gavage three times a week. ⁹ cfu/mL;

(2) High-fat diet group (HFD): Mice were fed a high-fat diet and water ad libitum, and were given 0.2 mL of PBS three times a week;

(3) Complete control group (ND) ingesting normal diet: Mice were given normal diet and water ad libitum, and 0.2 mL of PBS buffer (pH 7.2-7.4) was administered by gavage three times each time.

After 12 weeks of feeding, fresh feces of each mouse were collected on the 12th week of the experiment, the mice were sacrificed by cervical dislocation after collecting feces, and the liver, spleen and spleen were isolated and weighed. The distal small intestine and colon were partially embedded in paraffin and partially frozen in liquid nitrogen. The colon is a single segment, and the small intestine is divided into three segments. The intestinal canal is cut and flattened along the long axis. The number and size of intestinal adenomas in each segment are observed and recorded under a dissecting microscope. The distal small intestine and colon were rolled up and fixed along the horizontal axis, placed in 10% formalin, embedded in paraffin as soon as possible, and the remaining part was frozen in liquid nitrogen.

The results are shown in Figure 1a and Figure 1b, compared with the high-fat control group, the Dubosiella newyorkensis NYU-BL-A4 intervention group could significantly reduce the increase in liver weight induced by high-fat accumulation of fat, and could alleviate the inflammation-induced spleen. swollen.

Example 2: Dubosiella newyorkensis NYU-BL-A4 can significantly reduce the number of colonic adenomas

The grouping, modeling and treatment methods of experimental mice were the same as those in Example 1.

The results are shown in Figure 2. After different groups of mice received different dietary interventions for 12 weeks, the number of colon adenomas in each group of mice was counted under a dissecting microscope. The number of colonic adenomas in the high-fat diet alone group was significantly increased compared with the complete control group. Compared with the simple high-fat diet group, the number of colon adenomas in the Dubosiella newyorkensis NYU-BL-A4 intervention group was significantly reduced, indicating that the strain intervention can effectively reduce the number of colon adenomas and maintain intestinal homeostasis.

Example 3: Dubosiella newyorkensis NYU-BL-A4 significantly inhibits colon cancer cell proliferation

The cell experimental treatment method was the same as that in Example 1. In this example, two kinds of colon cancer cells, human colon adenocarcinoma cell line Caco-2 and human colon cancer cell line HCT116, were used, and the cells were treated with a medium containing Dunaliella, and the treatment conditions included different time gradients and concentration gradients.

Experimental cell culture:

(1) Caco-2 culture method: use MEM complete medium containing 20% fetal bovine serum FBS, 1% non-essential amino acids and 1% penicillin-streptomycin to culture cells at 37°C and 5% CO ₂ ;

(2) HCT116 culture method: DMEM medium containing 5% fetal bovine serum, 1% penicillin streptomycin was used to culture cells at 37°C and 5% CO ₂ .

The new medium was replaced every other day. When the cell confluence reached 70-80%, the Caco-2 cells were passaged at a ratio of 1:2 (the cells were seeded into two cell flasks for culture), and the HCT116 cells were passaged at a ratio of 1:3 (the cells were cultured in 1:3). were inoculated into 3 cell flasks for culture). Discard the old culture medium during subculture, add sterile PBS (phosphate buffered saline) to wash it twice, then add 0.25% (0.25g/100mL) trypsin to digest the suspended adherent cells, and inoculate an appropriate amount of cells into the culture Flasks continue to incubate or proceed to plating.

Intestinal cancer cell plating and processing:

(1) Two human intestinal cancer cell lines, Caco-2 and HCT116, were used, and the cells with a confluence of 70-80% were digested with trypsin and made into a cell suspension;

(2) After fully mixing, take 5 μL of cell suspension to a cell counting plate, and count the number of cells contained in 16 medium cells under a microscope; if 16 medium cells are known to contain 0.1 μL of cell suspension, the total number of cells in the suspension can be obtained ;

(3) Dilute the suspension with the corresponding medium to 2000 cells/100 μL, and after thorough mixing, seed the cells at 100 μL per well in a 96-well plate (a total of 36 wells around the 96-well plate do not add cell suspension, and each well adds 200 μL PBS as evaporation well);

(4) After the cells adhered completely, the old medium was discarded, and the PBS (phosphate buffered saline solution) solution containing Dubosieella newyorkensis NYU-BL-A4 and the blank control solution (PBS) were treated; the same treatment method Then use different processing time (24h, 48h, 72h) respectively.

Determination of the proliferation of intestinal cancer cells: With the help of electron coupling agent, WST-8 can be reduced to orange-yellow formazan by dehydrogenase in mitochondria. For the same cell, the shade of color is linearly related to the number of cells, and the more cells proliferate, the darker the color. The Cell Counting Kit-8 (CCK-8 kit) of Shanghai Biyuntian Company was used to detect the proliferation of colorectal cancer cells. The operations were as follows:

(1) After the treatment, discard the old medium, add 100 μL of basal medium without FBS and other additives to each well, and then add 10 μL of CCK8 solution, shake the plate gently to promote mixing;

(2) Put it back into the cell incubator for incubation, and use a microplate reader to measure the absorbance at 450 nm.

The results are shown in Figures 3a and 3b, the size of the absorbance OD ₄₅₀ can reflect the cell proliferation. The human colon adenocarcinoma cell line Caco-2 and the human colon cancer cell line HCT116 were cultured in a system containing Dunaliella, and Dunaliella could The growth of two intestinal cancer cell lines was significantly inhibited in a clear time-dependent manner.

Example 4: Regulation of Dunaliella NYU-BL-A4 on inflammatory factors that induce colon cancer

Extraction of total RNA from intestinal adenoma tissue: Take intestinal adenomas (2-4 polyp tissue blocks) from each group of APC ^Min+ mice and put them into 1.5 mL EP tubes without RNase, add 1 mL of Trizol to each tube, and add steel beads with high Fully grind with a flux tissue grinder, let stand for 10 min at room temperature, add 200 μL of chloroform, invert and mix for 30 s, and let stand for 5 min at room temperature. Centrifuge at 4°C for 15 min at 12000 rpm. Pipette the upper aqueous phase containing total RNA into a new RNase-free EP tube. Add 1 volume of isopropanol pre-cooled at 4°C to a new tube, and let stand at room temperature for 15 min. Centrifuge at 4°C for 15 min at 12,000 rpm to precipitate RNA and discard the supernatant. The precipitate was washed with 1 mL of pre-cooled 75% ethanol (DEPC water configuration), centrifuged at 12000 rpm and 4° C. for 10 min, the supernatant was discarded, and the washing step was repeated. Dry naturally at room temperature, evaporate the ethanol, dissolve the RNA precipitate with DEPC water; use a reverse transcription kit to synthesize cDNA for subsequent real-time fluorescence quantitative PCR.

SYBR Green method real-time fluorescent quantitative PCR:

Fluorescence quantitative PCR primers (synthesized by Shanghai Sangon Bioengineering Co., Ltd.):

GAPDH-F: AGG TCG GTG TGAACG GATTTG (SEQ ID NO. 1);

GAPDH-R: TGTAGA CCA TGTAGT TGA GGT CA (SEQ ID NO. 2);

Tnf-α-F: CAGGGCGGTGCCTATGTCTC (SEQ ID NO. 3);

Tnf-α-R: CGATCACCCCGAAGTTCAGTAG (SEQ ID NO. 4);

COX-2-F: ACGGTCCTGAACGCATTTATG (SEQ ID NO. 5);

COX-2-R: TTGGCCCCATTTAGCAATCTG (SEQ ID NO. 6).

Reaction system (10 μL system): 5 μL of 2×SYBR Green IMix, 0.5 μL of cDNA, 0.5 μL of upper and lower primers, and 4 μL of sterile water. The reaction system was added to a 96-well special reaction plate for real-time quantification, and four duplicate wells were set for each sample. Seal the 96-well plate with a special transparent film cover, centrifuge at 1500 rpm for 2 minutes, and put the reaction plate into the BIO RID fluorescence quantitative PCR instrument.

Reaction procedure: pre-denaturation: 95°C for 10 min; amplification: 95°C for 10s, 60°C for 20s, 72°C for 30s. 45 cycles; melting curve: 95°C for 5s, 65°C for 1min.

Calculate the relative expression of mRNA level: the Ct value of each sample when the fluorescence intensity reaches the threshold is measured by reaction, first subtract the Ct value of the internal reference gene (GAPDH) of the corresponding treatment group from the Ct value of each sample, and obtain the ΔCt value of each sample. For the same primer, the average ΔCt value of the control group was subtracted from the ΔCt value of each treatment group to obtain the ΔΔCt value of each sample. The relative expression level of mRNA in each sample = 2 ^{- ΔΔCt} .

The changes of inflammatory factors are closely related to the occurrence and development of intestinal tumors in Apcmin+ mice. The inflammatory response in the intestinal tumor of Apc ^min+ mice increased with the development and growth of the tumor, and the increased inflammatory response indicated that the tumor progressed faster and had a higher degree of malignancy. The results are shown in Figures 4a and 4b, Dubosiella newyorkensis NYU-BL-A4 can significantly inhibit the up-regulation of inflammatory factor gene expression levels such as Tnf-α and Cox2 in intestinal tumors of Apc ^min+ mice, indicating that Dubosiella newyorkensis reduces the The role of inflammation in colon cancer pathogenesis.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

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Claims (10)

1. Application of Dubosiella newyorkensis or a probiotic preparation containing Dubosiella in preparing a product for preventing, alleviating or improving colon cancer. 2. application according to claim 1, is characterized in that, described Dunaliella is NYU-BL-A4. 3 . The application according to claim 1 , wherein the probiotic preparation is used to improve the intestinal immunity of the body and inhibit the proliferation activity of intestinal cancer cells. 4 . 4. application according to claim 1, is characterized in that, in addition to Dunaliella in described probiotic preparation, also contains auxiliary material, described auxiliary material includes but not limited to excipient or food additive; Described Dunaliella in probiotic The content in the bacterial preparation is not less than 1.0×10 ⁷ cfu/mL or 1.0×10 ⁷ cfu/g. 5. The use according to claim 1, wherein the product comprises a drug or a pharmaceutical composition for use in at least one of (a) to (d): (a) relieve inflammation of the spleen; (b) reducing the number of colonic adenomas; (c) inhibit colon cancer cell proliferation; (d) Regulating the expression of inflammatory factors associated with colon cancer; The content of the Dunaliella in the medicine or pharmaceutical composition is not less than 1.0×10 ⁷ cfu/mL or 1.0×10 ⁷ cfu/g. 6. The use according to claim 5, wherein the inflammatory factor comprises Tnf-α, Cox2, IL-6 or interleukin-1β. 7. application according to claim 5, is characterized in that, described medicine or pharmaceutical composition also comprises pharmaceutically acceptable excipient; Described pharmaceutically acceptable excipient refers to any that can be used in the field of pharmacy diluents, adjuvants and/or carriers. 8. The application according to claim 1, wherein the products include but are not limited to food, health product drinks, enteral nutrition preparations, dietary supplements, veterinary drugs or feed additives. 9 . The application according to claim 8 , wherein the food, health product drink, enteral nutrition preparation, dietary supplement, veterinary medicine or feed additive also contains conventional auxiliary materials. 10 . 10. application according to claim 9 is characterized in that, described conventional adjuvant comprises a kind of in filler, corrigent, adhesive, disintegrating agent, lubricant, antacid and nutritional fortifier or more.

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