Thrombosis detection method in mice and the inhibitory effect of Lactobacillus plantarum on thrombosis

Abstract

The present invention provides a method for detecting mouse thrombosis and an inhibitory effect of plant lactobacillus on thrombosis, belonging to the field of thrombosis treatment, and the specific steps of the method for detecting mouse thrombosis are materials and reagents, instruments and equipment, animal experiments, four coagulation tests, serum and tissue inflammatory cytokine determination, tissue section detection, tissue mRNA expression detection, determination of microbial mRNA expression in mouse feces and statistical analysis. LP-KSFY04 of the present invention can effectively regulate the black tail degree and four coagulation items of thrombotic mice, and can also regulate inflammatory cytokines in serum, kidney and liver tissues, thereby exerting the anti-inflammatory effect of LP-KSFY04; further test results prove that LP-KSFY04 can regulate the mRNA expression of the NF-κB pathway, and avoid thrombosis blockage by regulation.

Description

Technical Field

[0001] The invention belongs to the field of thrombosis treatment, and particularly relates to a method for detecting mouse thrombosis and the inhibitory effect of Lactobacillus plantarum on thrombosis formation. Background Art

[0002] my country's Xinjiang region is vast, with a large number of high-quality pastures and developed animal husbandry. At the same time, there are many ethnic minorities in Xinjiang, and each ethnic minority has its own unique eating habits, but most of them have the habit of eating dairy products, including naturally fermented yogurt made by herders' families, which is also one of the most commonly consumed dairy products. Due to the unique geographical environment and natural fermentation methods in the areas where ethnic minority herders live in Xinjiang, compared with ordinary industrially produced yogurt on the market, the yogurt produced in the production process of Xinjiang's naturally fermented yogurt is more ecological and natural, and the microbial diversity is also richer. The nutritional value and health functions of yogurt are more prominent. In particular, the microorganisms in these naturally fermented dairy products have been naturally domesticated for thousands of years, have adapted to the living environment of Xinjiang, and have different fermentation characteristics and prebiotic functions, which are of great value for development and utilization.

[0003] Economic development has promoted the improvement of people's living standards, and has also changed people's dietary structure. The intake of a large amount of unhealthy food has caused a variety of chronic diseases, including cardiovascular and cerebrovascular diseases, which seriously threaten the health of middle-aged and elderly people. Most cardiovascular and cerebrovascular diseases have no obvious symptoms before the onset of the disease. Once the disease occurs suddenly and seriously, it is difficult to treat it in time, which seriously threatens the patient's life. Endogenous or exogenous damage will trigger a complex defense response, which will lead to an inflammatory response in the body. Inflammation is also an important factor in inducing thrombosis, and thrombosis will further aggravate inflammation. Thrombosis can cause certain damage to the body's organs. With the subsequent aggravation of inflammation and thrombosis, the damage to the body's organs will also increase. Severe thrombosis can cause complex inflammation, leading to dysfunction and tissue necrosis of multiple organs in the body, including the liver and kidneys. Under the inflammatory state, a large number of proinflammatory factors cause the blood to be in a hypercoagulable state, thereby causing the formation of thrombus; thrombus is also a factor that exacerbates the occurrence and development of inflammation in the body. The interaction between inflammation and thrombosis forms a complex network, causing serious damage to cardiovascular and cerebrovascular organs and tissues. Carrageenan can cause inflammatory reactions in experimental animals. After a large number of inflammatory factors appear in the animal's body, these factors will quickly enter the blood circulation, thereby causing damage or death of vascular endothelial cells and ultimately causing the formation of blood clots.

[0004] In the prior art, especially in experimental mice, the tail vessels are single-circulation vessels. After thrombosis occurs, blood cannot circulate through collateral circulation, so the tail tissue will gradually become ischemic, causing tissue necrosis.

[0005] However, the existing inhibitory effect on thrombosis has the problems of being unsuitable for various groups of people, poor thrombosis inhibition effect and unstable formation of Lactobacillus plantarum.

[0006] Therefore, it is very necessary to invent a method for detecting mouse thrombosis and the inhibitory effect of Lactobacillus plantarum on thrombosis. Summary of the invention

[0007] In order to solve the above technical problems, the present invention provides a method for detecting mouse thrombosis and the inhibitory effect of Lactobacillus plantarum on thrombosis, and the present invention is achieved through the following technical solutions:

[0008] The method for detecting thrombus in mice specifically comprises the following steps:

[0009] Step 1: Materials and reagents: Lactobacillus plantarum KSFY04 isolated from naturally fermented yogurt, SPF-grade male Kunming mice aged six weeks, weighing 23±2g; TNF-α, IL-6 and IL-1β detection kits; TRIzol reagent; SYBRGreenPCRMasterMix, qpCR primers; the rest of the reagents were domestic analytical grade;

[0010] Step 2: Instruments and equipment, PUN-2048A semi-automatic blood coagulation instrument, BX43 microscope, VarioskanLUX multifunctional microplate reader, StoponePlus quantitative PCR instrument;

[0011] Step 3: Animal experiment: mice were kept in an environment with a temperature of 20±1℃ and a humidity of 30% to 40%. The experimental mice were free to eat and drink water. The experiment officially started after the mice were adaptively fed for 7 days.

[0012] Step 4: Four coagulation tests: the collected mouse heart blood was tested using a semi-automatic coagulometer to measure activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB) and prothrombin time (PT);

[0013] Step 5: Serum and tissue inflammatory cytokine determination: The collected mouse heart blood was centrifuged at 1500rpm and 4°C for 10min, and the upper serum was removed for later use; at the same time, 0.1g of kidney and liver tissues were weighed, and then 0.9mL of normal saline was added to the kidney and liver tissues respectively, and then the tissues were homogenized at 4000rpm and 4°C for 10min, and the supernatant was collected after centrifugation for later use; finally, the levels of TNF-α, IL-6 and IL-1β inflammatory cytokines in serum and tissues were determined according to the detection kit method;

[0014] Step 6: Tissue section detection: After dissecting the mice, the kidney, liver and tail tissues of the mice were fixed with 10% formalin; after dehydration for 48 hours, the tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E); finally, the pathological changes of the tissues were observed with an optical microscope;

[0015] Step 7: Tissue mRNA expression detection, weigh 0.1g of mouse tail vein tissue, add 0.9mL of normal saline to the tissue for homogenization, then add 1.0mL of RNAzol to extract RNA from the mouse tail vein tissue; then observe;

[0016] Step 8: Determination of microbial mRNA expression in mouse feces: weigh 1.0 g of mouse feces, and determine the mRNA expression of microorganisms in mouse feces according to the tissue mRNA determination method in 1.3.5 to detect the composition of microorganisms in feces;

[0017] Step 9: Statistical analysis: All experiments were performed three times in parallel, and the results were expressed as the mean value, and the standard deviation was calculated. The experimental results were expressed as the mean ± standard deviation. At the same time, one-way analysis of variance was used to calculate whether there were significant differences between the data groups (P < 0.05).

[0018] Preferably, in step 3, 50 Kunming mice are randomly divided into 5 groups, each with 10 mice, namely a normal group, a model group, a drug positive control group, a LP-KSFY04 low concentration treatment (LP-KSFY04-L) group and a LP-KSFY04 high concentration treatment (LP-KSFY04-H) group; the mice in the normal group are intraperitoneally injected with a physiological saline solution at a dosage of 0.01 mL / g per day, and the mice in the other groups are intraperitoneally injected with a 0.2% concentration of carrageenan at a dosage of 0.01 mL / g per day. solution for 10 days; at the same time, the mice in the drug positive control group were gavaged with dipyridamole at a dosage of 20 mg / kg per day, and the mice in the LP-KSFY04-L group and LP-KSFY04-H group were gavaged with LP-KSFY04 at a dosage of 108 CFU / kg and 109 CFU / kg per day, and the gavage of dipyridamole and LP-KSFY04 also lasted for 10 days; after 10 days, the mice were killed by cervical dislocation, and the length of the black tail (thrombus) of the tail of each group of mice was recorded, and the heart blood and internal organs of the mice were dissected for testing.

[0019] Preferably, in step seven, the observation method adopts ultramicrospectrophotometry to measure the absorbance value of the extracted RNA at 260nm and 280nm, calculate the RNA purity and concentration, and adjust the RNA concentration to 1μg / μL; then, after generating cDNA by reverse transcription, prepare a reaction system containing 1μL cDNA, and the remaining reagents include 10μL SYBRGreen PCR Master Mix, 7μL sterile distilled water and 1μL upstream and downstream primer solutions; react in a quantitative PCR instrument, and the reaction conditions are 95℃ for 60s; 95℃ for 15s, 40 cycles; 55℃ for 30s; 72℃ for 35s; 95℃ for 30s; 55℃ for 35s.

[0020] The present invention also proposes the inhibitory effect of Lactobacillus plantarum on thrombosis in mice, mainly including that LP-KSFY04 can play the role of shortening the length of the black tail of thrombotic mice, just like dipyridamole as an antithrombotic drug; dipyridamole and LP-KSFY04 can play the role of controlling four coagulation indicators, which means that dipyridamole and LP-KSFY04 can play the role of inhibiting thrombosis; pathological observation also found that after thrombosis occurred in the tail of mice, inflammatory lesions also appeared in the kidney and liver tissues, which once again proved that the tail thrombosis of mice will involve kidney and liver lesions, and the drugs dipyridamole and LP-KSFY04 can inhibit these lesions, and LP-KSFY04 with probiotic potential can play an effect close to that of the drug dipyridamole; the drug dipyridamole has an intervention effect on the above inflammatory cytokines, thereby producing an inhibitory effect on thrombosis; at the same time, high concentrations of LP-KSFY04 also play a role The results showed that the two drugs had similar effects, and played a good intervention role in inflammatory cytokines and thrombosis; both dipyridamole and LP-KSFY04 could regulate the expression of NF-κB, ICAM-1, VCAM-1 and E-selectin, thereby exerting their possible good thrombosis inhibition effect; LP-KSFY04 could promote the production of more Bacteroidetes microorganisms and Bifidobacterium in the intestines of mice, while supplementing Lactobacillus and reducing Firmicutes microorganisms, thereby inhibiting the body's inflammation and promoting normal blood circulation by regulating intestinal microorganisms, and exerting the inhibitory effect of LP-KSFY04 on thrombosis; LP-KSFY04 had a good experimental thrombosis inhibition effect.

[0021] Compared with the prior art, the present invention has the following beneficial effects: Due to the inhibitory effect of the plant lactobacillus of mouse thrombosis on thrombosis, it is widely used in the field of thrombosis treatment technology. The LP-KSFY04 of the present invention can effectively regulate the black tail degree and four coagulation items of thrombotic mice, and can also regulate the inflammatory cytokines in serum, kidney and liver tissues, thereby exerting the anti-inflammatory effect of LP-KSFY04; further test results prove that LP-KSFY04 can regulate the mRNA expression of the NF-κB pathway, and avoid thrombosis blockage by regulation; at the same time, the present invention further shows that LP-KSFY04 can regulate the intestinal microbial composition of mice, making the intestinal microbial composition healthier, thereby maintaining the health of the body, reducing the degree of inflammation, and thus exerting the inhibitory effect of thrombosis; experimental results show that LP-KSFY04 has a good experimental thrombosis inhibitory effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a flow chart of the method for detecting thrombus in mice.

[0023] Figure 2 This is a schematic diagram of the black tail length of mouse thrombus.

[0024] Figure 3 Schematic diagram of H&E-stained sections of mouse tail (A), kidney (B), and liver (C) tissues.

[0025] Figure 4 This is a schematic diagram of mRNA expression in mouse tail vein tissue.

[0026] Figure 5 Schematic diagram of mRNA expression of Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacterium in mouse feces. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with the accompanying drawings:

[0028] In the figure:

[0029] As attached Figure 1 Shown

[0030] The detection of thrombus in mice includes the following steps:

[0031] S101: Materials and reagents, Lactobacillus plantarum KSFY04 isolated from naturally fermented yogurt, SPF-grade six-week-old male Kunming mice, weighing 23±2g; TNF-α, IL-6 and IL-1β detection kits; TRIzol reagent; SYBRGreenPCRMasterMix, qpCR primers; other reagents were domestic analytical grade;

[0032] S102: Instruments and equipment, PUN-2048A semi-automatic blood coagulation instrument, BX43 microscope, VarioskanLUX multifunctional microplate reader, StoponePlus quantitative PCR instrument;

[0033] S103: Animal experiment, mice were kept in an environment with a temperature of 20±1℃ and a humidity of 30%-40%. The experimental mice were free to eat and drink water. The experiment officially started after the mice were adaptively fed for 7 days;

[0034] S104: Four coagulation tests: the collected mouse heart blood was used to measure the activated partial thromboplastin time (APTT), thrombin time (TT), fibrinogen (FIB) and prothrombin time (PT) using a semi-automatic coagulometer;

[0035] S105: Serum and tissue inflammatory cytokine determination: the collected mouse heart blood was centrifuged at 1500rpm and 4°C for 10min, and the upper serum was removed for later use; at the same time, 0.1g of kidney and liver tissues were weighed, and then 0.9mL of normal saline was added to the kidney and liver tissues respectively, and then the tissues were homogenized at 4000rpm and 4°C for 10min, and the supernatant was collected after centrifugation for later use; finally, the levels of TNF-α, IL-6 and IL-1β inflammatory cytokines in serum and tissues were determined according to the detection kit method;

[0036] S106: tissue section detection, after the mice were dissected, the kidney, liver and tail tissues of the mice were fixed with 10% formalin; after dehydration for 48 hours, the tissue samples were embedded in paraffin, sectioned, and stained with hematoxylin-eosin (H&E); finally, the pathological changes of the tissues were observed with an optical microscope;

[0037] S107: Tissue mRNA expression detection, weigh 0.1g of mouse tail vein tissue, add 0.9mL of normal saline to the tissue for homogenization, then add 1.0mL of RNAzol to extract RNA in the mouse tail vein tissue; then observe;

[0038] S108: Determination of microbial mRNA expression in mouse feces: weigh 1.0 g of mouse feces, and determine the mRNA expression of microorganisms in mouse feces according to the tissue mRNA determination method in 1.3.5 to detect the composition of microorganisms in feces;

[0039] S109: Statistical analysis: All experiments were performed in triplicate, and the results were expressed as mean values, and the standard deviation was calculated. The experimental results were expressed as mean ± standard deviation. At the same time, one-way analysis of variance was used to calculate whether there were significant differences between the data groups (P < 0.05).

[0040] Preferably, in S103, 50 Kunming mice are randomly divided into 5 groups, each with 10 mice, namely a normal group, a model group, a drug positive control group, a LP-KSFY04 low concentration treatment (LP-KSFY04-L) group and a LP-KSFY04 high concentration treatment (LP-KSFY04-H) group; the mice in the normal group are intraperitoneally injected with a saline solution at a dosage of 0.01 mL / g per day, and the mice in the other groups are intraperitoneally injected with a 0.2% carrageenan solution at a dosage of 0.01 mL / g per day for 10 days; at the same time, the mice in the drug positive control group are intravenously administered with dipyridamole at a dosage of 20 mg / kg per day. The mice in the LP-KSFY04-L group and the LP-KSFY04-H group were gavaged with LP-KSFY04 at a dosage of 108 CFU / kg and 109 CFU / kg daily. The gavage of dipyridamole and LP-KSFY04 also lasted for 10 days. After 10 days, the mice were killed by cervical dislocation, and the length of the black tail (thrombus) of the tail of each group of mice was recorded. At the same time, the mice were dissected and the heart blood and internal organs were taken for testing.

[0041] Preferably, in S107, the observation method uses ultramicrospectrophotometry to measure the absorbance value of the extracted RNA at 260nm and 280nm, calculate the RNA purity and concentration, and adjust the RNA concentration to 1μg / μL; then, after generating cDNA by reverse transcription, prepare a reaction system containing 1μL cDNA, and the remaining reagents include 10μL SYBR Green PCR Master Mix, 7μL sterile distilled water and 1μL upstream and downstream primer solutions; react in a quantitative PCR instrument, and the reaction conditions are 95℃ for 60s; 95℃ for 15s, 40 cycles; 55℃ for 30s; 72℃ for 35s; 95℃ for 30s; 55℃ for 35s.

[0042] The present invention also proposes the inhibitory effect of Lactobacillus plantarum on thrombosis in mice, mainly including that LP-KSFY04 can play the role of shortening the length of the black tail of thrombotic mice, just like dipyridamole as an antithrombotic drug; dipyridamole and LP-KSFY04 can play the role of controlling four coagulation indicators, which means that dipyridamole and LP-KSFY04 can play the role of inhibiting thrombosis; pathological observation also found that after thrombosis occurred in the tail of mice, inflammatory lesions also appeared in the kidney and liver tissues, which once again proved that the tail thrombosis of mice will involve kidney and liver lesions, and the drugs dipyridamole and LP-KSFY04 can inhibit these lesions, and LP-KSFY04 with probiotic potential can play an effect close to that of the drug dipyridamole; the drug dipyridamole has an intervention effect on the above inflammatory cytokines, thereby producing an inhibitory effect on thrombosis; at the same time, high concentrations of LP-KSFY04 also play a role The results showed that the two drugs had similar effects, and played a good intervention role in inflammatory cytokines and thrombosis; both dipyridamole and LP-KSFY04 could regulate the expression of NF-κB, ICAM-1, VCAM-1 and E-selectin, thereby exerting their possible good thrombosis inhibition effect; LP-KSFY04 could promote the production of more Bacteroidetes microorganisms and Bifidobacterium in the intestines of mice, while supplementing Lactobacillus and reducing Firmicutes microorganisms, thereby inhibiting the body's inflammation and promoting normal blood circulation by regulating intestinal microorganisms, and exerting the inhibitory effect of LP-KSFY04 on thrombosis; LP-KSFY04 had a good experimental thrombosis inhibition effect.

[0043] Specific implementation examples

[0044] Tissue mRNA expression detection:

[0045] 0.1 g of mouse tail vein tissue was weighed, 0.9 mL of normal saline was added to the tissue for homogenization, and then 1.0 mL of RNAzol was added to extract RNA from the mouse tail vein tissue; then the absorbance of the extracted RNA was measured at 260 nm and 280 nm using ultramicrospectrophotometry, the RNA purity and concentration were calculated, and the RNA concentration was adjusted to 1 μg / μL; then cDNA was generated by reverse transcription, and a reaction system containing 1 μL of cDNA was prepared, and the remaining reagents included 10 μL SYBR Green PCR Master Mix, 7 μL of sterile distilled water, and 1 μL of primer solution for upstream and downstream; the reaction was carried out in a quantitative PCR instrument, and the reaction conditions were 95°C for 60 s; 95°C for 15 s, 40 cycles; 55°C for 30 s; 72°C for 35 s; 95°C for 30 s; 55°C for 35 s; GAPDH was selected as an internal reference (Table 1), and its related genes were analyzed by the 2-ΔΔCt method;

[0046] Table 1 Primer sequences used in this experiment

[0047]

[0048] Mouse black tail length

[0049] After intraperitoneal injection of carrageenan, black tails appeared at the tail tips of mice in each group, indicating the presence of tail thrombosis. The black tails of mice in the model group were the longest (8.43±0.52cm), which was significantly longer than those in the other groups (P<0.05). The black tails of mice in the LP-KSFY04-H group were shorter, at 3.02±0.44cm, only longer than those in the dipyridamole group (2.35±0.47cm). The black tails of the LP-KSFY04-H and dipyridamole groups were significantly shorter than those of the LP-KSFY04-L group (5.89±0.58cm, P<0.05). Figure 2 As shown:

[0050] Figure 2 Mouse thrombus black tail length

[0051] Different letters indicate significant differences between the groups at the P < 0.05 level, and the same letters indicate no significant differences;

[0052] Mouse coagulation test

[0053] The APTT of the normal group mice was significantly higher than that of the other groups (P<0.05, Table 2), while the TT, FIB and PT were significantly lower than those of the other groups (P<0.05); the model group showed the opposite situation, with the APTT significantly lower than that of the other groups, while the TT, FIB and PT were significantly higher than those of the other groups (P<0.05); the APTT of the LP-KSFY04-H group was significantly higher than that of the LP-KSFY04-L group, and the TT, FIB and PT were significantly lower than those of the LP-KSFY04-L group (P<0.05), and the APTT, TT, FIB and PT of the LP-KSFY04-H group were close to those of the dipyridamole group, with no significant difference (P>0.05);

[0054] Table 2 Indices of four coagulation items in mice

[0055]

[0056] Inflammatory cytokines in mouse serum, kidney tissue and liver tissue

[0057] As shown in Tables 3, 4 and 5, the levels of TNF-α, IL-6 and IL-1β in the serum, kidney tissue and liver tissue of the mice in the normal group, dipyridamole group, LP-KSFY04-H group, LP-KSFY04-L group and model group showed a trend from low to high; among them, the levels of TNF-α, IL-6 and IL-1β in the dipyridamole group and LP-KSFY04-H group were significantly (P<0.05) lower than those in the LP-KSFY04-L group, but there was no significant difference between the dipyridamole group and the LP-KSFY04-H group (P>0.05);

[0058] Table 3 Serum TNF-α, IL-6 and IL-1β levels in mice

[0059]

[0060]

[0061] Table 4 Levels of TNF-α, IL-6 and IL-1β in mouse kidney tissue

[0062]

[0063] Table 5 TNF-α, IL-6 and IL-1β levels in mouse liver tissue

[0064]

[0065] Pathological observation of mouse tail tissue, kidney tissue and liver tissue

[0066] Depend on Figure 3As shown in the figure, the caudal vein vascular morphology of the normal group mice was intact and round, and the vascular wall morphology was smooth; the caudal vein vascular wall of the model group mice had significant inflammatory exudation and leukocyte infiltration, bleeding around the blood vessels, and thrombosis in the blood vessels; LP-KSFY04 and dipyridamole could both alleviate the lesions of the caudal vein vascular of mice, and the effects of LP-KSFY04-H and dipyridamole were better than LP-KSFY04-L, and the effects of LP-KSFY04-H and dipyridamole were similar; observation of kidney tissue also found that the kidney tissue of the model group mice had lesions, irregular glomerular morphology, some even ruptured, and severe inter-tissue inflammatory cell infiltration; the kidney tissue and cells of the normal group mice were intact, and LP-K Both SFY04-H and dipyridamole can reduce the inflammatory lesions of renal tissue caused by thrombosis, making the morphology of renal tissue close to that of the normal group; at the same time, LP-KSFY04-L can also reduce the lesions of renal tissue in thrombotic mice to a certain extent; observation of H&E stained sections of liver tissue found that the liver tissue of mice in the normal group was intact, the cells were uniform in size, the central vein was round, the liver lobules and hepatocytes were intact, and the cell boundaries were clear; the central veins of the liver tissue of mice in the model group were irregular in shape, with a large number of inflammatory cells infiltrating, the boundaries of hepatocytes were chaotic, and the nuclei of some hepatocytes were ruptured; LP-KSFY04-H and dipyridamole can significantly inhibit the damage of hepatocytes caused by thrombosis, with similar effects and better than the effect of LP-KSFY04-L;

[0067] Figure 3 H&E stained sections of mouse tail (A), kidney (B), and liver (C) tissues

[0068] mRNA expression in mouse tail vein tissue

[0069] like Figure 4 As shown, the expression of NF-κBp65, ICAM-1, VCAM-1, E-selectin, IL-6 and TNF-α mRNA in the tail vein of mice in the model group was the strongest; LP-KSFY04-L, LP-KSFY04-H and dipyridamole could significantly (P<0.05) down-regulate the expression of NF-κBp65, ICAM-1, VCAM-1, E-selectin, IL-6 and TNF-α in the tail vein of thrombotic mice; and there was no significant difference in these expressions between the LP-KSFY04-H and dipyridamole groups, which were higher than those in the normal group and lower than those in the model group and LP-KSFY04-L group.

[0070] Figure 4 mRNA expression in mouse tail vein tissue mRNA expression in mouse tail vein tissue

[0071] like Figure 5As shown, the mRNA expression of Firmicutes microorganisms in the feces of mice in the normal group was significantly lower (P<0.05) than that of other groups, while the mRNA expression of Bacteroidetes and Bifidobacterium was significantly higher (P<0.05) than that of other groups; after thrombosis induction, the mRNA expression of Firmicutes microorganisms in the feces of mice in the model group was the highest, while the expression of Bacteroidetes, Lactobacillus and Bifidobacterium was the lowest; LP-KSFY04 and dipyridamole could reduce the expression of Firmicutes in the feces of mice with thrombosis, and enhance the expression of Bacteroidetes and Bifidobacterium; in addition, there was no significant difference in the expression of Lactobacillus between the normal group and the LP-KSFY04 group, which was significantly higher than that of the other groups (P<0.05);

[0072] Figure 5 mRNA expression of Firmicutes, Bacteroidetes, Lactobacillus, and Bifidobacterium in mouse feces

[0073] In summary, the present invention observed the newly discovered Lactobacillus plantarum LP-KSFY04, and observed the inhibitory effect of this Lactobacillus plantarum on mouse thrombosis through animal experiments; the experimental results showed that LP-KSFY04 can effectively regulate the black tail degree and four coagulation items of thrombotic mice, and can also regulate the inflammatory cytokines in serum, kidney and liver tissues, thereby exerting the anti-inflammatory effect of LP-KSFY04; further test results proved that LP-KSFY04 can regulate the mRNA expression of the NF-κB pathway, and avoid thrombosis blockage by regulation; at the same time, the present invention further shows that LP-KSFY04 can regulate the intestinal microbial composition of mice, making the intestinal microbial composition healthier, thereby maintaining the health of the body and reducing the degree of inflammation, thereby exerting the inhibitory effect of thrombosis; the experimental results show that LP-KSFY04 has a good experimental thrombosis inhibitory effect; however, the present invention still lacks the results of human trials, so further research needs to strengthen clinical research.

[0074] Utilizing the technical solution described in the present invention, or those skilled in the art designing a similar technical solution inspired by the technical solution of the present invention to achieve the above-mentioned technical effects, all fall within the protection scope of the present invention.

Claims

1. A method for detecting the inhibitory effect of Lactobacillus plantarum KSFY04 on mouse thrombosis, characterized in that: The detection method specifically includes the following steps: Step 1: Materials and reagents: Lactobacillus plantarum KSFY04 isolated from naturally fermented yogurt, SPF grade six-week-old male Kunming mice, weighing 23±2g; TNF-a, IL-6 and IL-1β detection kits; TRIzol reagent; SYBR Green PCR Master Mix; qpCR primers; other reagents were all domestic analytical grade; Step 2: Instruments and equipment, PUN-2048A semi-automatic blood coagulation instrument, BX43 microscope, VarioskanLUX multifunctional microplate reader, StoponePlus quantitative PCR instrument; Step 3: Animal experiment: mice were kept in an environment with a temperature of 20±1℃ and a humidity of 30% to 40%. The experimental mice were free to eat and drink water. The experiment officially started after the mice were adaptively fed for 7 days. Step 4: Four coagulation tests: the collected mouse heart blood was used to measure the activated partial thromboplastin time APTT, thrombin time TT, fibrinogen FIB and prothrombin time PT using a semi-automatic coagulometer; Step 5: Serum and tissue inflammatory cytokine determination: The collected mouse heart blood was centrifuged at 1500rpm and 4°C for 10min, and the upper serum was removed for later use; at the same time, 0.1g of kidney and liver tissues were weighed, and then 0.9mL of normal saline was added to the kidney and liver tissues respectively, and then the tissues were homogenized at 4000rpm and 4°C for 10min, and the supernatant was collected after centrifugation for later use; finally, the levels of TNF-a, IL-6 and IL-1β inflammatory cytokines in serum and tissues were determined according to the detection kit method; Step 6: Tissue section detection: After dissecting the mice, fix the kidney, liver and tail tissues of the mice with 10% formalin; after dehydration for 48 hours, embed the tissue samples in paraffin, slice them, and stain them with hematoxylin-eosin; finally, observe the pathological changes of the tissues with an optical microscope; Step 7: Tissue mRNA expression detection, weigh 0.1g of mouse tail vein tissue, add 0.9mL of normal saline to the tissue for homogenization, then add 1.0mL of RNAzol to extract RNA in the mouse tail vein tissue; then observe; Step 8: Determination of microbial mRNA expression in mouse feces. Weigh 1.0 g of mouse feces and determine the mRNA expression of microorganisms in mouse feces to detect The composition of fecal microorganisms; Step 9: Statistical analysis: All experiments were performed in triplicate, and the results were expressed as the mean value, and the standard deviation was calculated. The experimental results were expressed as the mean ± standard deviation. At the same time, one-way analysis of variance was used to calculate whether there was a significant difference between the data groups P < 0.05; In step 3, 50 Kunming mice were randomly divided into 5 groups, each with 10 mice, namely a normal group, a model group, a drug positive control group, a LP-KSFY04 low concentration treatment LP-KSFY04-L group and a LP-KSFY04 high concentration treatment LP-KSFY04-H group; the mice in the normal group were intraperitoneally injected with a physiological saline solution at a dosage of 0.01 mL / g per day, and the mice in the other groups were intraperitoneally injected with a 0.2% carrageenan solution at a dosage of 0.01 mL / g per day. liquid for 10 days; at the same time, the mice in the drug positive control group were gavaged with dipyridamole at a dosage of 20 mg / kg per day, and the mice in the LP-KSFY04-L group and LP-KSFY04-H group were gavaged with LP-KSFY04 at a dosage of 108 CFU / k and 109 CFU / kg per day, and the gavage of dipyridamole and LP-KSFY04 also lasted for 10 days; after 10 days, the mice were killed by cervical dislocation, and the black tail length of the mice in each group was recorded, and the heart blood and internal organs of the mice were dissected for testing.

2. The detection method according to claim 1, characterized in that In step seven, the observation method uses ultramicrospectrophotometry to measure the absorbance value of the extracted RNA at 260nm and 280nm, calculate the RNA purity and concentration, and adjust the RNA concentration to 1μg / μL; then, after generating cDNA by reverse transcription, prepare a reaction system containing 1μL cDNA, and the remaining reagents include 10μL SYBR Green PCR Master Mix, 7μL sterile distilled water and 1μL upstream and downstream primer solutions; react in a quantitative PCR instrument, and the reaction conditions are 95°C for 60s, 95°C for 15s and 40 cycles, 55°C for 30s, 72°C for 35s, 95°C for 30s, and 55°C for 35s.

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