CN115300522A - Application of Weissella cibaria D-2 exopolysaccharide in inhibition of human colon cancer cells - Google Patents
Application of Weissella cibaria D-2 exopolysaccharide in inhibition of human colon cancer cells Download PDFInfo
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Abstract
The invention relates to the technical field of colon cancer treatment and discloses application of Weissella cibaria (Weissellacia) D-2 extracellular polysaccharide in inhibition of human colon cancer cells. The Weissella sinus-feeding bacteria (preservation number: CCTCC M20221190) is screened out from healthy infant feces, extracellular polysaccharide of the Weissella sinus-feeding bacteria promotes the apoptosis process of colon cancer cells through a Fas/Fasl-Caspase8-Caspase3 pathway, and powerful evidence is provided for the anti-tumor effect of the Weissella sinus-feeding bacteria extracellular polysaccharide. The Weissella sinus D-2 exopolysaccharide acts on human colon cancer cells, and is found to effectively play an anti-colon cancer role through a cell apoptosis promoting mode, and the Weissella sinus D-2 exopolysaccharide is a cell component of symbiotic microorganisms in intestinal tracts of mammals, so that the Weissella sinus D-2 exopolysaccharide is safer than other anti-tumor drugs from the perspective of clinical application.
Description
Technical Field
The invention relates to the technical field of colon cancer treatment, in particular to application of Weissella cibaria D-2 extracellular polysaccharide in inhibition of human colon cancer cells.
Background
Colorectal cancer (CRC) is the third most common malignancy in the world and is also the second leading cause of cancer-related death. Most patients with CRC have middle and late stage diseases, and seriously threaten the life and health of human beings in China and all over the world. Meanwhile, chemotherapy and targeted therapy are accompanied by toxic and side effects which vary from person to person and are sometimes even life-threatening. Therefore, it is of great interest to find more effective treatments for CRC. Numerous studies have shown that intestinal microorganisms are closely associated with the development and development of colorectal cancer. Several lactobacilli, enterococci and streptococci and their metabolites have been reported to play an important role in the prevention and treatment of cancer.
Weissella cibaria (Weissella cibaria) is a species of lactic acid bacteria that is present in the intestinal tract, participates in the composition of the intestinal microecology, and is in direct contact with the intestinal barrier. Intestinal microbiology sequencing results showed that weissella civora decreased in colorectal cancer patients. In addition, researches show that Weissella has the effects of regulating immunity and enhancing anti-tumor effect, but whether Weissella fossa can directly inhibit colorectal cancer and related mechanisms are not clear. In addition, because of the high requirement of the storage environment of live probiotics, certain specific strains have the possibility of opportunistic infection, so that the live bacteria have certain limitation and risk in clinical application. Due to the above problems in current clinical medicine, the research on the new mechanism of effective anti-cancer products of probiotics against colon cancer and the development of new intervention modes and drugs based on the mechanism are urgent.
The extracellular polysaccharide produced by lactobacillus has the functions of regulating immunity, resisting inflammation and resisting tumor. At present, the research on Weissella cibaria extracellular polysaccharide mainly focuses on food fermentation, and the report on the anti-tumor effect and the related mechanism of the Weissella cibaria extracellular polysaccharide is not seen at present. Therefore, it is of great importance to find Weissella cibaria having an anti-colorectal cancer effect, and to discover effective metabolites, and to develop new in vivo biopharmaceuticals based thereon. Disclosure of Invention
Technical problem to be solved
The Weissella cibaria strain (Weissella Cibaria D-2) is screened from healthy infant feces, and the preservation number of the Weissella cibaria strain is CCTCC M20221190. The extracellular polysaccharide of the strain promotes the apoptosis process through a Fas/Fasl-Caspase8-Caspase3 pathway, and provides strong evidence for the anti-tumor effect of the Weissella civora extracellular polysaccharide.
(II) technical scheme
In order to verify that the Weissella cibaria extracellular polysaccharide has the functions of resisting proliferation, migration invasion, apoptosis induction and cycle retardation on colon cancer cells, the invention provides the following technical scheme: the application of Weissella cibaria D-2 exopolysaccharide in inhibiting human colon cancer cells is characterized by comprising the following two application methods:
1) Weissella civora D-2 exopolysaccharide for inhibiting human colon cancer cells
(1) Selection and grouping of cells: selecting human colon cancer cells HT29 and SW480, grouping the selected cells, setting the selected cells as a control group and a Weissella sinus D-2 extracellular polysaccharide experimental group, wherein the culture environment temperature is 37 ℃, and the culture environment gas components are five percent of carbon dioxide and ninety-five percent of air;
(2) And (3) cell survival rate detection: paving the human colon cancer cells HT29 and SW480 cultured in the steps in a pore plate with the number of 96, adding 10 mu L of CCK8 solution into each pore of the pore plate, then putting the pore plate into an incubator for incubation, and measuring the absorbance at 450nm after incubating for 2-4h in the incubator;
(3) And (3) detecting cell apoptosis: paving human colon cancer cells HT29 and SW480 in a pore plate with the pore number of 6, digesting the cells, collecting the cells, respectively adding 5 mu L of Annexin V dye and 5 mu L of PI dye, adding 5 mu L of Annexin V dye, incubating for 15 minutes by light, adding 5 mu L of PI dye, inoculating for 5 minutes in a dark place, transferring the inoculated cells to a flow tube, and detecting the apoptosis condition of the cells by using a flow cytometer within one hour;
(4) Molecular mechanism exploration: paving the human colon cancer cells HT29 and SW480 in a pore plate with the pore number of 6, respectively cracking and collecting the cells by RIPA and Trizol, and exploring a related molecular mechanism for the Weissella sinus D-2 extracellular polysaccharide to play a role by a WB method;
2) Weissella cibaria D-2 exopolysaccharide for treating HT-29 tumor-bearing nude mice
(1) Molding HT-29 tumor-bearing nude mice: after HT-29 passage of human colon cancer cells under aseptic operation environment, the cells are stably cultured for twenty-four hours, the cell concentration is adjusted to 5 x 106 cells/mL in logarithmic growth phase, and the cells are injected into the right axillary subcutaneous tissue of a nude mouse at the cell suspension concentration of 100 mu L/mouse, and the operation is completed within 1 hour. A blank control group, a Weissella sinus D-2 exopolysaccharide low-dose group, a Weissella sinus D-2 exopolysaccharide medium-dose group and a Weissella sinus D-2 exopolysaccharide high-dose group are arranged.
(2) Administration: the medicine is administrated 1 time every 3 days for 4 times in total, and the intratumoral injection amount of each time is 0.1mL/20g of body mass. The doses and routes of administration were as follows:
blank control group: healthy nude mice were injected intratumorally with PBS (0.1 mL/20g physical mass/day) each time, and were free to eat and drink water;
weissella cibaria D-2 exopolysaccharide low dose group: inoculating colon cancer cells HT-29 nude mice, injecting Weissella cibaria D-2 exopolysaccharide (50 mg/kg body mass/day) into tumor each time, and freely taking and drinking water;
weissella civora D-2 exopolysaccharide medium dose group: inoculating colon cancer cells HT-29 nude mice, injecting Weissella cibaria D-2 exopolysaccharide (100 mg/kg physical quantity/day) intratumorally, and freely eating and drinking water;
weissella civora D-2 exopolysaccharide high dose group: inoculating colon cancer cells HT-29 nude mice, injecting Weissella cibaria D-2 exopolysaccharide (150 mg/kg body mass/day) into tumor each time, and freely eating and drinking water;
(3) Tumor volume measurement: during the fourteen day feeding period, tumor volume measurements were taken daily and calculated as follows:
tumor volume (mm) 3 )＝(L×S 2 )/2
In the formula: l is the tumor mass major diameter; s is the tumor mass minor axis;
(4) Detection of body weight and organ condition: nude mice were weighed daily during the fourteen day feeding period. On the fifteenth day, the mice are sacrificed by cervical dislocation, the transplanted tumor and the heart, liver, spleen, lung and kidney are taken out, HE staining is carried out on the heart, liver, spleen, lung and kidney, and the condition of tissue injury is observed;
(5) Molecular mechanism exploration: the WB method of the tumor tissues stripped from each group verifies the relevant molecular mechanism of the action of Weissella sinus D-2 extracellular polysaccharide.
Preferably, the selection of the cells and the control group in the group are placed in a culture medium for culture, the culture time is seventy-two hours, the selection of the cells and the cells of the Weissella sinus D-2 extracellular polysaccharide experimental group in the group are placed in a Weissella sinus D-2 extracellular polysaccharide solution with the concentration of 0.7mg/mL for culture, and the culture time is also seventy-two hours.
Preferably, the cell viability = 100% absorbance of cells at a concentration of exopolysaccharide treated/control during the cell viability assay.
Preferably, said Weissella sinus D-2 is a strain isolated and identified from the feces of infants.
(III) advantageous effects
Compared with the prior art, the invention provides the application of Weissella cibaria D-2 exopolysaccharide in inhibiting human colon cancer cells, and the application has the following beneficial effects:
the Weissella sinus D-2 exopolysaccharide is applied to inhibition of human colon cancer cells, by acting the Weissella sinus D-2 exopolysaccharide on the human colon cancer cells, the Weissella sinus D-2 exopolysaccharide is found to effectively play an anti-colon cancer role through a necrotic apoptosis mode, the Weissella sinus D-2 exopolysaccharide can inhibit the human colon cancer cells in vivo and in vitro through inducing apoptosis, the Weissella sinus D-2 exopolysaccharide is a natural component existing in the intestinal tracts of mammals, and compared with other anti-tumor drugs, the Weissella D-2 exopolysaccharide is safer from the perspective of clinical application, and the Weissella sinus D-2 exopolysaccharide in the research can resist gastrointestinal fluid stress and cannot influence the application effect due to oral administration.
Drawings
FIG. 1 is a graph of the effect of Weissella D-2 exopolysaccharides on cell proliferation;
FIG. 2 is a graph of the effect of Weissella D-2 exopolysaccharides on apoptosis;
FIG. 3 is a graph of the effect of Weissella D-2 exopolysaccharides on protein expression in tumor cells;
FIG. 4 is a graph showing the effect of Weissella furstriiformis D-2 exopolysaccharides on tumors, body weights and organs of nude mice.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
The first embodiment is as follows: weissella civora D-2 exopolysaccharide inhibits human colon cancer cells:
(1) Selection and grouping of cells: selecting human colon cancer cells HT29 and SW480, grouping the selected cells, setting the selected cells as a control group and a Weissella sinus D-2 extracellular polysaccharide experimental group, wherein the culture environment temperature is 37 ℃, and the culture environment gas components are five percent of carbon dioxide and ninety-five percent of air;
(2) And (3) cell survival rate detection: paving the human colon cancer cells HT29 and SW480 cultured in the steps in a pore plate with the number of 96, adding 10 mu of LCCK8 solution into each pore of the pore plate, then putting the pore plate into an incubator for incubation, and measuring the absorbance at 450nm after incubating for 2-4h in the incubator;
(3) And (3) detecting cell apoptosis: paving human colon cancer cells HT29 and SW480 in a pore plate with the pore number of 6, digesting the cells, collecting the cells, respectively adding 5 mu L of Annexin V dye and 5 mu L of LPI dye, adding 5 mu L of Annexin V dye, incubating for 15 minutes by light, adding 5 mu L of PI dye, inoculating for 5 minutes in a dark place, transferring the inoculated cells to a flow tube, and detecting the apoptosis condition of the cells by using a flow cytometer within one hour;
(4) Molecular mechanism exploration: spreading human colon cancer cells HT29 and SW480 in a pore plate with the number of pores of 6, respectively cracking by RIPA and Trizol to collect cells, and exploring a related molecular mechanism for the Weissella sinus D-2 extracellular polysaccharide to play a role by a WB method.
The following conclusions can be drawn through the implementation steps:
A. the IC50 of SW480 and HT29 after 72h of the action of the Weissella cibaria D-2 exopolysaccharides is respectively about 0.52mg/mL and 0.58mg/mL;
B. the Weissella cibaria D-2 extracellular polysaccharide can promote the apoptosis of SW480 cells of human colon cancer, although the amplification is not large, the proportion of total apoptotic cells is only increased by 1.67 percent, but the effect is more stable, and the statistical significance is achieved; the apoptosis promoting effect of the D-2-exopolysaccharide on the human colon cancer cell HT29 is more obvious than that of the human colon cancer cell SW480, and the proportion of total apoptotic cells is increased by 6.61%. The results show that the Weissella cibaria D-2 exopolysaccharide can promote the apoptosis of colon cancer cells;
C. the water average of Fas, fasL, cleared-Caspase 8 and cleared-Caspase 3 expression proteins in Weissella sinus D-2 extracellular polysaccharide treated cells is increased, which indicates that Weissella sinus D-2 extracellular polysaccharide can induce colon cancer cell apoptosis, thereby inhibiting the growth of colon cancer cells.
Example two: weissella cibaria D-2 exopolysaccharide for treating HT-29 tumor-bearing nude mice:
(1) Molding HT-29 tumor-bearing nude mice: after HT-29 passage of human colon cancer cells under aseptic operation environment, the cells are stably cultured for twenty-four hours, the cell concentration is adjusted to 5 x 106 cells/mL in logarithmic growth phase, and the cells are injected into the right axillary subcutaneous tissue of a nude mouse at the cell suspension concentration of 100 mu L/mouse, and the operation is completed within 1 hour. A blank control group, a Weissella sinus D-2 exopolysaccharide low-dose group, a Weissella sinus D-2 exopolysaccharide medium-dose group and a Weissella sinus D-2 exopolysaccharide high-dose group are arranged.
(2) Administration: the medicine is administrated 1 time every 3 days for 4 times, and the intratumoral injection amount is 0.1mL/20g of body mass. The doses and routes of administration were as follows:
blank control group: healthy nude mice were injected intratumorally with PBS (0.1 mL/20g physical mass/day) each time, and were free to eat and drink water;
weissella cibaria D-2 exopolysaccharide low dose group: inoculating colon cancer cells HT-29 nude mice, injecting Weissella cibaria D-2 exopolysaccharide (50 mg/kg physical quantity/day) intratumorally, and freely eating and drinking water;
weissella civora D-2 extracellular polysaccharide medium dose group: inoculating colon cancer cells HT-29 nude mice, injecting Weissella cibaria D-2 exopolysaccharide (100 mg/kg body mass/day) into tumor each time, and freely eating and drinking water;
weissella civora D-2 exopolysaccharide high dose group: inoculating colon cancer cells HT-29 nude mice, injecting Weissella cibaria D-2 exopolysaccharide (150 mg/kg body mass/day) into tumor each time, and freely eating and drinking water;
(3) Tumor volume measurement: during the fourteen day feeding period, tumor volume measurements were taken daily and calculated as follows:
tumor volume (mm) 3 )＝(L×S 2 )/2
In the formula: l is the tumor mass major diameter; s is the tumor mass minor axis;
(4) Detection of body weight and organ condition: nude mice were weighed daily during the fourteen day feeding period. On the fifteenth day, the mice are sacrificed by cervical dislocation, the transplanted tumor and the heart, liver, spleen, lung and kidney are taken out, HE staining is carried out on the heart, liver, spleen, lung and kidney, and the condition of tissue injury is observed;
(5) Molecular mechanism exploration: the WB method is used for verifying the related molecular mechanism of the Weissella sinus D-2 extracellular polysaccharide
The following conclusions can be drawn through the implementation steps:
A. the tumor volumes of tumor-bearing nude mice in the Weissella fossilis D-2 extracellular polysaccharide treatment group are lower than those of the control group, which indicates that the Weissella fossilis D-2 extracellular polysaccharide can inhibit the growth of tumors or tumor cells;
B. the body weight and organ tissues of the nude mice with tumor in the Weissella sinus D-2 exopolysaccharide treatment group have no obvious difference from those in the control group, which shows that the Weissella sinus D-2 exopolysaccharide has no obvious toxic or side effect on the body weight and important organs of the nude mice and has certain safety guarantee;
C. compared with a control group, the protein levels of Fas, fasL, clear-Caspase 8 and clear-Caspase 3 in tumor tissues of nude mice with tumor in the Weissella sinus D-2 extracellular polysaccharide treated group are increased, which shows that Weissella D-2 extracellular polysaccharide can induce apoptosis of colon tumor, thereby inhibiting colon cancer.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (4)
1. The application of Weissella cibaria D-2 exopolysaccharide in inhibiting human colon cancer cells is characterized by comprising the following two application methods:
1) Weissella civora D-2 exopolysaccharide inhibits human colon cancer cells:
(1) Selection and grouping of cells: selecting human colon cancer cells HT29 and SW480, grouping the selected cells, setting the selected cells as a control group and a Weissella sinus D-2 extracellular polysaccharide experimental group, wherein the culture environment temperature is 37 ℃, and the culture environment gas components are five percent of carbon dioxide and ninety-five percent of air;
(2) Cell survival rate detection: paving the human colon cancer cells HT29 and SW480 cultured in the steps in a pore plate with the number of 96, adding 10 mu L of CCK8 solution into each pore of the pore plate, then putting the pore plate into an incubator for incubation, and measuring the absorbance at 450nm after incubating for 2-4h in the incubator;
(3) And (3) detecting cell apoptosis: paving human colon cancer cells HT29 and SW480 in a pore plate with the pore number of 6, digesting the cells, collecting the cells, respectively adding 5 mu L of Annexin V dye and 5 mu L of PI dye, adding 5 mu L of Annexin V dye, incubating for 15 minutes by light, adding 5 mu L of PI dye, inoculating for 5 minutes in a dark place, transferring the inoculated cells to a flow tube, and detecting the apoptosis condition of the cells by using a flow cytometer within one hour;
(4) Molecular mechanism exploration: paving human colon cancer cells HT29 and SW480 in a pore plate with the number of pores of 6, respectively cracking and collecting the cells by RIPA and Trizol, and exploring a related molecular mechanism for the Weissella cibaria D-2 extracellular polysaccharide to play a role by a WB method;
2) Weissella cibaria D-2 exopolysaccharide for treating HT-29 tumor-bearing nude mice
(1) Molding HT-29 tumor-bearing nude mice: after HT-29 passage of human colon cancer cells under aseptic operation environment, the cells are stably cultured for twenty-four hours, the cell concentration is adjusted to 5 x 106 cells/mL in logarithmic growth phase, and the cells are injected into the right axillary subcutaneous tissue of a nude mouse at the cell suspension concentration of 100 mu L/mouse, and the operation is completed within 1 hour. Setting a blank control group, a Weissella sinus-eating D-2 exopolysaccharide low-dose group, a Weissella sinus-eating D-2 exopolysaccharide medium-dose group and a Weissella sinus-eating D-2 exopolysaccharide high-dose group.
(2) Administration: the medicine is administrated 1 time every 3 days for 4 times, and the intratumoral injection amount is 0.1mL/20g of body mass. The doses and routes of administration were as follows:
blank control group: healthy nude mice were injected intratumorally with PBS (0.1 mL/20g physical mass/day) each time, and were free to eat and drink water;
weissella cibaria D-2 exopolysaccharide low dose group: inoculating colon cancer cells HT-29 nude mice, injecting Weissella cibaria D-2 exopolysaccharide (50 mg/kg body mass/day) into tumor each time, and freely taking and drinking water;
weissella civora D-2 exopolysaccharide medium dose group: inoculating colon cancer cells HT-29 nude mice, injecting Weissella cibaria D-2 exopolysaccharide (100 mg/kg body mass/day) into tumor each time, and freely eating and drinking water;
weissella civora D-2 exopolysaccharide high dose group: inoculating colon cancer cells HT-29 nude mice, injecting Weissella cibaria D-2 exopolysaccharide (150 mg/kg body mass/day) into tumor each time, and freely eating and drinking water;
(3) Tumor volume measurement: during the fourteen day feeding period, tumor volume measurements were taken daily and calculated as follows:
tumor volume (mm) 3 )＝(L×S 2 )/2
In the formula: l is the tumor mass major diameter; s is the tumor mass minor diameter;
(4) Detection of body weight and organ condition: nude mice were weighed daily during the fourteen day feeding period. On the fifteenth day, the mice are sacrificed by cervical dislocation, the transplanted tumor and the heart, liver, spleen, lung and kidney are taken out, HE staining is carried out on the heart, liver, spleen, lung and kidney, and the condition of tissue injury is observed;
(5) Molecular mechanism exploration: the WB method of the tumor tissues stripped from each group verifies the relevant molecular mechanism of the action of Weissella sinus D-2 extracellular polysaccharide.
2. The use of weissella sinus D-2 exopolysaccharide in inhibiting human colon cancer cells as claimed in claim 1, wherein the selection of cells and the control group in the group are cultured in a culture medium for seventy two hours, and the selection of cells and the cells in the weissella sinus D-2 exopolysaccharide experimental group in the group are cultured in a weissella sinus D-2 exopolysaccharide solution with a concentration of 0.7mg/mL for seventy two hours.
3. The use of weissella D-2 exopolysaccharides in the inhibition of human colon cancer cells according to claim 1, wherein the cell viability assay is cell viability = 100% absorbance of exopolysaccharide treated cells/control cells at a concentration.
4. The use of weissella sinus D-2 exopolysaccharides in the inhibition of human colon cancer cells according to claim 1, characterized in that said weissella sinus D-2 is a strain identified by isolation from infant feces.
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| CN105441357A (en) * | 2015-12-16 | 2016-03-30 | 北京工商大学 | Lactobacillus plantarum for producing antitumor activity exopolysaccharides |
| CN113164528A (en) * | 2018-12-10 | 2021-07-23 | 韩国食品研究院 | Pharmaceutical composition for preventing or treating cancer comprising Weissella cibaria WIKIM28 as an active ingredient |
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| CN105441357A (en) * | 2015-12-16 | 2016-03-30 | 北京工商大学 | Lactobacillus plantarum for producing antitumor activity exopolysaccharides |
| CN113164528A (en) * | 2018-12-10 | 2021-07-23 | 韩国食品研究院 | Pharmaceutical composition for preventing or treating cancer comprising Weissella cibaria WIKIM28 as an active ingredient |
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| CN116426415A (en) * | 2023-03-09 | 2023-07-14 | 四川大学 | Weissella sinica TE2103 and application thereof |
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