Six probiotics for preventing severe heatstroke and application thereof

Abstract

The invention discloses six probiotics for preventing severe heatstroke and application of the six probiotics. A probiotic composition is prepared from bacillus coagulans XY2, lactobacillus plantarum KL100, lactobacillus rhamnosus TR08, lactobacillus helveticus TR05, lactobacillus casei TR01 and bifidobacterium longum TR17. The invention discloses novel application of the probiotic composition in prevention of severe heatstroke, heatstroke combined with liver injury, enhancement of intestinal mucosal barrier function, heatstroke combined with lung injury and heatstroke combined with myocardial injury. The sextuple probiotic preparation can effectively protect the state of an organism, enhance the resistance of the organism to high temperature, maintain the balance of a body temperature regulation mechanism, and prevent or relieve the phenomenon of severe heatstroke of the organism, and has the effects of preventing severe heatstroke and relieving heatstroke combined liver injury, lung injury and myocardial injury. Meanwhile, the six probiotic preparations can effectively enhance the intestinal mucosal barrier, and a new method and thought are provided for preventing the adverse outcome of extreme high temperature and complications thereof.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of hexavalent probiotics in the preparation of drugs for preventing severe heatstroke, heatstroke combined with liver damage, enhancing intestinal mucosal barrier function, heatstroke combined with lung damage, and heatstroke combined with myocardial damage. Background Art

[0002] The liver plays a vital role in metabolism, crucial for maintaining and regulating lipid and glucose levels and energy metabolism. Extreme heat can cause liver swelling, vacuolization, steatosis, and extensive centrilobular necrosis. Among the multi-organ damage caused by heatstroke, acute liver injury and its progression to acute liver failure are the leading causes of death in heatstroke patients. Heat exposure can also disrupt the structure and function of intestinal cells, increasing intestinal mucosal permeability and allowing intestinal bacteria and endotoxins to enter the bloodstream. The intestinal mucosal barrier, the core of the intestinal defense system, is primarily composed of four components: a mechanical barrier (tightly connected epithelial cells), a chemical barrier (secretions such as mucins and antimicrobial peptides), an immune barrier (including immune cells and their secreted cytokines), and a biological barrier (intestinal microbiota and their metabolites). These barriers work synergistically to maintain a stable intestinal environment and protect against the invasion of external harmful substances. The link between intestinal and liver diseases, and the complex interactions between the intestine, liver, and microbiome, is known as the "gut-liver axis" theory. Liver metabolic processes and disease progression are closely linked to the intestinal microbiome. The liver can influence the composition and function of the intestinal microbiome through secretion of bile acids and immunoglobulin A. Currently, there is no effective treatment for heatstroke combined with acute liver injury, so finding effective preventive interventions is an urgent issue with important public health implications.

[0003] For the lungs, extreme heat often causes acute lung injury in the form of pulmonary edema and acute respiratory distress syndrome. Pathological manifestations include inflammation, oxidative stress, and pulmonary dysfunction, characterized by diffuse alveolar damage to the alveolar epithelium. The intestine and lungs have long been considered closely connected organs. "The lungs and large intestine are exterior and interior to each other" is a key concept in Traditional Chinese Medicine's holistic approach, dating back to the Yellow Emperor's Classic of Internal Medicine, which states, "The lungs are connected to the large intestine, and the large intestine is the organ of conduction." From a histological and embryological perspective, the embryonic development of lung and intestine tissues is homologous. Both originate from the primitive foregut, and their cell morphology, proliferation, and apoptosis are similar during early embryonic development. The microbiome and immune systems of the intestine and lung are interconnected and interactive, both in healthy and pathological states, hence the term "gut-lung axis." Currently, there is a lack of effective treatments for acute lung injury associated with heatstroke, making the development of effective preventive interventions a top priority and of significant public health significance.

[0004] Extreme high temperatures can significantly increase the burden on the cardiovascular system, induce or aggravate heart disease, and even lead to sudden death. Studies have found that heat waves are significantly associated with increased mortality from myocardial infarction. When the intestinal barrier is damaged, bacterial endotoxins (such as LPS) enter the blood, triggering low-grade inflammation throughout the body, promoting arteriosclerosis and myocardial damage. The two-way interaction between the intestine and the heart involves multiple levels such as intestinal flora, metabolites, immune inflammation, and neural regulation, and is called the "gut-heart axis." Currently, there is a lack of effective treatments for acute myocardial injury complicated by heatstroke, and "heart-gut interaction" may become a new target for the prevention and treatment of cardiovascular diseases induced by extreme high temperatures.

[0005] Probiotics play an important role in treating immune and inflammatory diseases by improving intestinal barrier function. Furthermore, probiotic intervention can improve metabolic disorders by regulating the homeostasis of the intestinal flora. However, there are no technical insights or literature reports on the use of hexavalent probiotics for preventing severe heatstroke or heatstroke-related acute liver, lung, or myocardial damage. Summary of the Invention

[0006] In view of the problems existing in the prior art, the present invention aims to provide a technical solution for preventing severe heatstroke by providing a six-linked probiotic and its application. The present invention is specifically implemented through the following technical solutions:

[0007] A six-in-one probiotic for preventing severe heatstroke, comprising: Bacillus coagulans XY2, Lactobacillus plantarum KL100, Lactobacillus rhamnosus TR08, Lactobacillus helveticus TR05, Lactobacillus casei TR01, and Bifidobacterium longum TR17, wherein the probiotics are mixed in a ratio of 1:1:1:1:1:1:1 in terms of live bacteria count.

[0008] The use of the above-mentioned six-linked probiotics in the preparation of medicines for preventing and treating severe heatstroke.

[0009] The use of the above-mentioned six-combination probiotics in the preparation of enhancing the intestinal mucosal barrier function caused by severe heatstroke.

[0010] The use of the above-mentioned six-linked probiotics in the preparation of drugs for preventing severe heatstroke combined with liver damage.

[0011] Furthermore, heat stroke combined with liver damage is heat stroke combined with acute liver damage.

[0012] The use of the above-mentioned six-combination probiotics in the preparation of drugs for preventing severe heatstroke combined with lung injury.

[0013] Furthermore, the heat stroke combined with lung injury is heat stroke combined with acute lung injury.

[0014] The use of the above-mentioned six-linked probiotics in the preparation of drugs for preventing severe heatstroke combined with myocardial injury.

[0015] Furthermore, the heat stroke combined with myocardial injury is heat stroke combined with acute myocardial injury.

[0016] The dosage form of the above-mentioned six-in-one probiotics is a liquid oral dosage form, the solvent is an aqueous solution or milk, and the water temperature is controlled below 37°C. The specific preparation steps are: mix the six-in-one probiotics freeze-dried powder and the aqueous solution or milk evenly, and the water temperature is below 37°C. Uniform means that the system is in a uniform phase.

[0017] The dosage of this six-in-one probiotic is 0.065g / mL calculated based on the final concentration in mice, and it contains approximately 195 million CFU live probiotics.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] (1) The six-linked probiotics provided by the present invention are proposed for the first time as a new application in the field of preparing a product for preventing severe heatstroke / heatstroke combined with liver damage.

[0020] (2) The present invention clearly demonstrates that the six-linked probiotic preparation has excellent efficacy in preventing severe heatstroke, significantly improving the body's internal environmental homeostasis, increasing the body's tolerance to high temperatures through multiple mechanisms, and mitigating tissue damage caused by heat exposure. This preparation can effectively maintain the physiological balance of the body's core temperature regulation system and reduce the risk of severe heatstroke.

[0021] (3) At the same time, the six-linked probiotic preparation can effectively protect the body from or alleviate acute liver damage associated with heatstroke and prevent heat stress-related liver dysfunction, making it a potential drug for the prevention and treatment of heatstroke-related liver damage. The dual protective effect of six-linked probiotics in preventing severe heatstroke and related liver damage provides a new intervention strategy for the prevention and treatment of heat stress-related diseases.

[0022] (4) The six-linked probiotic preparation enhances the intestinal mucosal barrier function, strengthens the body's ability to resist high temperatures, and effectively prevents harmful substances from entering the blood circulation through the mucosal barrier. The technical content provided by this invention has important application value in the field of drug research and development for severe heatstroke and its complications. The breakthrough progress of this technology has opened up new research directions for the clinical prevention and treatment of heat-related diseases and drug development.

[0023] (5) The six-linked probiotic preparation can effectively protect the body from or alleviate acute lung injury associated with heatstroke and prevent heat stress-related lung function abnormalities, and can be used as a potential drug for the prevention and treatment of heatstroke-related lung injury. The dual protective effect of the six-linked probiotic in preventing severe heatstroke and its associated lung injury provides a new intervention strategy for the prevention and treatment of heat stress-related diseases.

[0024] (6) At the same time, the six-linked probiotic preparation can effectively protect the body from or alleviate acute myocardial damage associated with heat stroke, prevent heat stress-related myocardial dysfunction, and can be used as a potential drug for the prevention and treatment of heat stroke-related myocardial damage. The dual protective effect of six-linked probiotics in preventing severe heat stroke and related myocardial damage provides a new intervention strategy for the prevention and treatment of heat stress-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a graph showing the effect of the six-combination probiotic preparation of the present invention on the core body temperature changes of mice;

[0026] Figure 2 Graph showing the effects of the six-combination probiotic preparation of the present invention on mouse liver morphology, serum alanine aminotransferase (ALT), and mouse serum aspartate aminotransferase (AST);

[0027] Figure 3 Graph showing the effect of the six-combination probiotic preparation of the present invention on phosphatidic acid phosphohydrolase (Lipin 1) in mouse liver;

[0028] Figure 4 This figure shows the effect of the six-combination probiotic preparation of the present invention on the intestinal mucosal barrier of mice;

[0029] Figure 5 This figure shows the effect of the six-combination probiotic preparation of the present invention on the lung morphology of mice;

[0030] Figure 6 This figure shows the effect of the six-combination probiotic preparation of the present invention on lung inflammation in mice;

[0031] Figure 7 This figure shows the effect of the six-combination probiotic preparation of the present invention on oxidative stress in the lungs of mice;

[0032] Figure 8 This figure shows the effect of the six-combination probiotic preparation of the present invention on the pulmonary vascular barrier function of mice;

[0033] Figure 9 Graph showing the effects of the six-combination probiotic preparation of the present invention on mouse heart morphology, lactate dehydrogenase (LDH), and creatine kinase isoenzyme (CK-MB);

[0034] Figure 10 This figure shows the effect of the six-combination probiotic preparation of the present invention on the cardiac function of mice;

[0035] Figure 11 This figure shows the effect of the six-combination probiotic preparation of the present invention on cardiac inflammation in mice;

[0036] Figure 12 The effect of the six-combination probiotic preparation of the present invention on cardiac oxidative stress in mice (Note: Compared with the NC group, * P<0.05,** P<0.01, *** P<0.001; compared with the HC group, # P<0.05,

[0037] ## P<0.01, ### P < 0.001. The t test was used to evaluate the significant differences, and the results are expressed as mean ± SEM. DETAILED DESCRIPTION

[0038] The present invention will be further described below with reference to specific embodiments.

[0039] Each of the six probiotics in the present invention is of existing technology and can be directly obtained by purchase, among which:

[0040] Bacillus coagulans XY2, its deposit number is CGMCC NO.33934, and the deposit date is March 24, 2025.

[0041] Lactobacillus plantarum KL100, its preservation number is CGMCC NO.27192, and the preservation date is April 23, 2023.

[0042] Lactobacillus rhamnosus TR08, its deposit number is CCTCC NO: M2016548, and its deposit date is October 10, 2016.

[0043] Lactobacillus helveticus TR05: its deposit number is CCTCC NO: M2016569, and the deposit date is October 17, 2016.

[0044] Lactobacillus casei TR01: its preservation number is CCTCC NO: M2016567, and the preservation date is October 17, 2016.

[0045] Bifidobacterium longum TR17: its deposit number is CCTCC NO: M2017265, and the deposit date is May 15, 2017.

[0046] Example 1: Preparation of Six-linked Probiotic Solution and Model Intervention Plan

[0047] The preparation of the six-linked probiotic solution of the present invention is twice a day according to the oral dosage of people, 2g per bag, and the daily dosage of 70kg of standard body weight is 4g (4g / 70kg). If calculated by the dosage per unit body weight, the equivalent dose of mice (standard body weight 20g) is equivalent to 9.1 times that of humans. The dosage of mice per day obtained based on this conversion factor is 0.52g / kg, i.e. 0.52mg / g, and the gavage volume according to mice is 8μL / g. The dosage of the six-linked probiotics in the oral agent is 0.065mg / μL (i.e. 0.065g / mL) based on the final concentration of mice, i.e. 1mL aqueous solution (below 37°C) requires 0.065g of probiotic freeze-dried powder. According to the actual weight of the mice and the gavage volume, the gavage volume of each mouse is obtained, and the maximum gavage volume does not exceed 200μL. The probiotic content finally injected is about 195 million CFU viable bacteria. In the process of optimizing the dosage of the six-linked probiotics, anything that can achieve the technical effects described in the present invention belongs to the technical solution described in the present invention and falls within the scope of protection of the present invention.

[0048] Model intervention plan:

[0049] Healthy male C57BL / 6N mice, 8 weeks old, were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. (license number: SCXK(Zhe)2019-0001). They were housed at the Experimental Animal Center of Zhejiang Chinese Medical University at 22±1°C on a 12 / 12 hour day / night cycle and were given free access to food and water. After one week of acclimatization in an SPF animal facility, the C57BL / 6 mice were randomly divided into four groups of six mice each. The groupings were as follows:

[0050] 1) Normal control (NC)

[0051] 2) Preventive probiotic intervention control group (Control+Probiotics, CP)

[0052] 3) Extreme high temperature exposure group (Heat control, HC)

[0053] 4) Extreme heat exposure + preventive probiotic intervention group (Heat+Probiotics, HP)

[0054] One week before the start of heat exposure, mice were given probiotics once daily by gavage at 9:00 PM. A control group received an equal amount of ddH2O based on mouse body weight. HC and HP groups were exposed to a high temperature of 39.5±0.5°C. During this period, mice were deprived of food and water, and their core body temperatures were monitored using a rectal thermometer. Exposure was terminated when the core body temperature of the HC group reached its maximum or when heat shock occurred. HP and HC groups were exposed for the same duration. Following exposure, both groups were placed in a 22±1°C environment for nine hours of recovery, with water and food provided.

[0055] Research plan: ① Based on the construction of the animal model in Example 1, the mice recovered to 9 hours after high temperature exposure, were anesthetized with intraperitoneal injection of sodium pentobarbital, and blood was collected from the hypothalamic vena cava to measure ALT and AST. The mice in the control group were given the same treatment except for high temperature exposure. ② Detection of gene expression levels in liver tissue: Using the above-mentioned animal model, the total RNA of the liver sample was extracted using RNAiso Plus reagent, the RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit, and the mRNA level of Lipin 1 was detected using SYBR Green. ③ Detection of protein expression levels in liver tissue: Using the above-mentioned animal model, the total protein of the liver sample was extracted, the concentration was determined, and the protein level of Lipin 1 was detected by Western Blot. ④ The levels of serum LPS, DAO and D-Lactate were detected by ELISA. ⑤ Detection of gene expression levels in lung tissue: Using the above animal model, total RNA of lung samples was extracted using RNAiso Plus reagent, and RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit. SYBR Green was used to detect the expression of IL-6, Nf-κb, Cat, Sod1, Hmox-1, and Nqo1. 、 ⑥ Based on the construction of the animal model in Example 1, the mice were anesthetized by intraperitoneal injection of sodium pentobarbital for 9 hours after recovery from high temperature exposure, and blood was collected from the hypothalamic vena cava to measure CK, LDH and CK-MB. The control group mice were given the same treatment except for high temperature exposure. ⑦ Echocardiography was used to evaluate the cardiac function of the mice. ⑧ Detection of gene expression levels in cardiac tissue: Using the above-mentioned animal model, RNAiso Plus reagent was used to extract total RNA from lung samples, and the RNA was reverse transcribed into cDNA using a cDNA reverse transcription kit, and SYBR Green was used to detect the mRNA levels of IL-6 and HO-1.

[0056] Example 2: Effect of the Six-linked Probiotic Preparation on Core Body Temperature Changes in Mice

[0057] Figure 1The effects of the six-combination probiotic formulation of the present invention on core body temperature changes in mice were analyzed. The results showed that when the HC group mice reached a state of heat shock (i.e., their core body temperature reached its maximum), under the same conditions and exposure time, the core body temperatures of both the HP and HC groups were significantly elevated compared to the NC group. The highest core body temperature of the HP group mice was significantly lower than that of the HC group mice (39.8°C vs. 41.2°C), indicating that probiotics can prevent excessive core body temperature increases during heat exposure. Excessive core body temperature can lead to heat exhaustion or even death in mice. When removed from the high-temperature environment, the core body temperatures of the heat-exposed mice began to drop. The HC group mice all experienced a rapid and significant drop, dropping below 30°C within a short period of time, indicating temperature dysregulation. When the body temperature is too low, the mice become unconscious, sluggish, and even experience shock, coma, and death. When the core body temperature of the HC group mice reached its lowest point, the lowest core body temperature in the HP group was significantly higher than that in the HC group (32.5°C vs. 25.5°C). The core body temperature of the HP group mice decreased significantly less than that of the HC group mice. When the temperature dropped to the lowest temperature, it was still within the normal temperature regulation range of the body. No serious body temperature disorder occurred. No symptoms related to severe heat stroke occurred. Figure 1 A). In the recovery period, the core body temperature of mice in the HP group returned to normal levels (35.1°C), while the core body temperature of mice in the HC group (27.2°C) did not return to normal levels ( Figure 1 B).

[0058] The above results demonstrate that the six-linked probiotic preparation can effectively regulate the dynamic changes in body temperature in mice exposed to high temperatures. Specifically, it inhibits the rapid rise in body temperature during heat exposure, significantly slows the rate of core temperature rise, and enhances the body's resistance to heat; it promotes the restoration of body temperature homeostasis and accelerates thermoregulation during the recovery period. The six-linked probiotic preparation also significantly alleviates the decrease in core temperature during the recovery period in heat-exposed mice, quickly restoring the mice's core temperature and preventing severe temperature dysregulation in heat-exposed mice. When a mouse's core temperature drops below normal, beyond the range that the body can regulate, it can affect metabolism, the central nervous system, the circulatory system, and liver function, and in severe cases, can be life-threatening. Therefore, the experimental results demonstrate that the six-linked probiotic preparation can prevent severe heatstroke in mice, improve their ability to adapt to heat, reduce the incidence of severe heatstroke, and improve physiological function during the recovery period. Therefore, the use of the six-linked probiotic preparation in the preparation of a drug for the prevention of severe heatstroke is expected to be effective in preventing severe heatstroke and improving the overall functional status of patients recovering from heat exposure.

[0059] Example 3: Effects of the six-combination probiotic preparation on mouse liver morphology, serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST)

[0060] From the liver tissue morphology and staining of the four groups of mice, namely NC, CP, HC and HP, it can be seen that the livers of the mice in the NC and CP groups were pink or crimson with smooth surfaces; the livers of the mice in the HC group were darker in color and dark red in tissue, with irregular and disordered surface structures. The livers of the mice in the HP group were similar in color to those in the NC group. The liver cells of the mice in the NC and CP groups were arranged radially in a strip-like pattern with the central vein as the center, forming hepatocyte cords, and the irregular cavities between the hepatocyte cords were sinusoids; the liver tissue morphology of the mice in the HC group was abnormal, with congestion in the sinusoids and vacuolar changes. Preventive probiotic intervention can significantly improve the above phenomena ( Figure 2 A).

[0061] ALT is mainly present in the cytoplasm of liver cells. Under normal circumstances, its content in serum is very low. When liver cells are damaged, the permeability of cell membranes increases, and ALT in cells will be released into the blood. ALT is the most sensitive detection indicator of liver function damage. A low serum ALT level means that liver function has not been seriously damaged. Compared with the control group, the serum ALT of mice in the heat exposure group was significantly increased, and the preventive probiotic intervention can significantly reduce the serum ALT level ( Figure 2 B).

[0062] Most AST is present in the mitochondria of the liver. Under normal circumstances, its content in the serum is very low. When severe liver damage occurs, it is usually accompanied by mitochondrial membrane damage, and AST will be released into the blood. AST reflects the severity of liver cell damage. A low serum AST level means that liver function is not significantly damaged. Similarly, compared with the control group, the serum AST of mice in the heat exposure group was significantly increased, and preventive probiotic intervention can significantly reduce the level of serum AST ( Figure 2 C). The above results indicate that preventive probiotic intervention can significantly improve liver morphology and liver damage in mice exposed to extreme high temperature.

[0063] Example 4: Effect of the Six-linked Probiotic Preparation on Lipin1 in Mouse Liver

[0064] The experimental results showed that compared with the control group, the mRNA expression level of Lipin 1 in the liver of mice in the heat exposure group was significantly decreased. After the intervention of preventive probiotics, the mRNA expression of Lipin 1 could be increased to a certain extent ( Figure 3 A). Compared with the NC group, the protein expression level of Lipin 1 in the liver of mice in the HC group was significantly decreased; compared with the HC group, the protein expression level of Lipin 1 in the liver of mice in the HP group was significantly increased ( Figure 3BC). The Lipin 1 gene was identified in 2001 as a key gene closely associated with lipid deposition. The protein it encodes belongs to the Lipin family. In the cytoplasm, Lipin 1 catalyzes the dephosphorylation of phosphatidic acid to diacylglycerol through its phosphatidic acid phosphohydrolase activity. This process is a key step in triglyceride synthesis and is crucial for lipid storage. Downregulation of Lipin 1 expression leads to elevated diacylglycerol levels in the liver, thereby interfering with the insulin receptor signaling pathway, ultimately leading to lipid metabolism disorders and lipid deposition. The above experimental results show that Lipin 1 expression in the liver of mice exposed to high temperatures was significantly downregulated, suggesting a disorder of hepatic lipid metabolism. At the same time, compared with the heat-exposed group, Lipin 1 expression in the liver of mice exposed to probiotics was significantly increased. These results suggest that preventive probiotic intervention can significantly enhance Lipin 1 expression in the liver of mice exposed to extreme heat and improve hepatic lipid metabolism.

[0065] Example 5: Effects of the Six-Combination Probiotic Preparation on the Intestinal Mucosal Barrier

[0066] Compared with the control group, the serum D-Lactate, LPS and DAO levels of mice in the heat exposure group were significantly increased; compared with the heat exposure group, the serum D-Lactate of mice exposed to extreme high temperature after preventive probiotic intervention was significantly decreased ( Figure 4 A), LPS levels showed a downward trend ( Figure 4 B), DAO levels decreased significantly ( Figure 4C). Levels of D-lactic acid (D-Lactate), endotoxin (LPS), and diamine oxidase (DAO) are important indicators of intestinal barrier integrity. DAO is primarily derived from intestinal mucosal epithelial cells. Elevated levels generally reflect structural damage to intestinal epithelial cells and functional disruption of the intestinal mucosal mechanical barrier. As a major metabolic product of the intestinal resident flora, elevated D-Lactate levels serve as a hallmark indicator of impaired intestinal mucosal integrity and increased permeability. LPS, a key component of the outer membrane of Gram-negative bacteria, is typically concentrated in the intestine. As the largest source of endotoxins in the body, LPS is not easily released into the bloodstream when the intestinal barrier is intact. However, when the intestinal barrier is compromised, LPS can penetrate the intestinal mucosa and enter the bloodstream, triggering endotoxemia and systemic inflammation. These experimental results indicate that serum D-Lactate, LPS, and DAO levels in mice exposed to high temperatures were significantly elevated, indicating impaired intestinal mucosal integrity and increased permeability, leading to the release of endotoxins into the bloodstream and causing systemic inflammation. At the same time, compared with the high temperature exposure group, under the action of probiotics, the D-Lactate, LPS and DAO related to the mucosal barrier function of mice were significantly reduced. This shows that preventive probiotic intervention significantly enhances the intestinal mucosal barrier function of mice exposed to extreme high temperatures.

[0067] Example 6: Effects of the Six-Combination Probiotic Preparation on Lung Morphology in Mice

[0068] Compared with the control group, a large number of alveolar collapses occurred in the lungs of heat-exposed mice, and the alveolar surface area was significantly reduced, suggesting that heat exposure caused atelectasis. Compared with the heat-exposed group, preventive probiotic intervention significantly alleviated the alveolar collapse in the lungs of heat-exposed mice, and the alveolar surface area was significantly increased ( Figure 5 A). Semi-quantitative results showed that the alveolar area / total area in the lungs of heat-exposed mice was significantly lower than that in the control group; while the alveolar area / total area in the lungs of mice treated with preventive probiotics was significantly higher than that in the heat-exposed group. The above results indicate that probiotic intervention can effectively alleviate atelectasis caused by heat exposure ( Figure 5 B).

[0069] Example 7: Effect of the Six-linked Probiotic Preparation on Lung Inflammation in Mice

[0070] Compared with the control group, the mRNA levels of inflammatory factors IL-6 and NF-κb in the lungs of heat-exposed mice were significantly upregulated; compared with the heat-exposed group, preventive probiotic intervention significantly inhibited the mRNA levels of IL-6 ( Figure 6 A) and Nf-κb ( Figure 6 B) mRNA levels. This indicates that preventive probiotic intervention can significantly improve lung inflammation induced by heat exposure in mice.

[0071] Example 8: Effects of the Six-Combination Probiotic Preparation on Oxidative Stress in Mouse Lungs

[0072] The detection of the expression levels of antioxidant enzymes related to oxidative stress showed that the mRNA levels of antioxidant enzymes Cat, Sod1, Hmox-1 and Nqo1 in the lungs of heat-exposed mice were significantly higher than those in the control group; compared with the heat-exposed group, the preventive probiotic intervention significantly downregulated the expression of Cat( Figure 7 A), Sod1( Figure 7 B), Homx-1( Figure 7 C) and Nqo1( Figure 7 D) mRNA levels. Cat, Sod1, Hmox-1, and Nqo1 are all key antioxidant enzymes that synergistically maintain cellular redox homeostasis and protect against oxidative stress damage by decomposing hydrogen peroxide, scavenging superoxide, and degrading pro-oxidant heme and reducing quinones, respectively. These results suggest that heat exposure disrupts lung redox homeostasis in mice, leading to compensatory upregulation of antioxidant enzyme expression; preventive probiotic intervention can alleviate heat exposure-induced redox imbalance and restore antioxidant enzyme levels.

[0073] Example 9: Effects of the Six-Combination Probiotic Preparation on Pulmonary Vascular Barrier Function in Mice

[0074] Compared with the control group, the mRNA levels of pulmonary vascular barrier-related channel proteins Occludin and Claudin-4 in heat-exposed mice were significantly increased, and the mRNA level of Claudin-5 was significantly decreased; compared with the heat-exposed group, preventive probiotic intervention significantly reduced the expression of Occludin ( Figure 8 A), Claudin-4 ( Figure 8 B) mRNA level, and increased the mRNA level of Claudin-5 ( Figure 8 C). The tight junction proteins occludin, claudin-4, and claudin-5, primarily participate in the formation and regulation of tight junctions within the pulmonary vascular barrier (PVB), jointly maintaining the integrity and permeability of the alveolar epithelium and ensuring normal gas exchange and fluid balance in the lungs. Lung injury leads to decreased expression of the core tight junction protein claudin-5. The body may upregulate the expression of occludin and claudin-4 to repair damaged tight junctions and maintain PVB integrity. These results suggest that high temperature exposure can disrupt the tight junction structure of the PVB in mice, impairing the "molecular sieve" function of the endothelial barrier and leading to upregulation of occludin and claudin-4 expression, thereby repairing the damaged barrier. Preventive probiotic intervention can upregulate claudin-5 expression, alleviate inflammation and oxidative stress, protect PVB integrity, and restore normal expression levels of occludin and claudin-4.

[0075] Example 10: Effects of the six-combination probiotic preparation on cardiac morphology, lactate dehydrogenase (LDH) and creatine kinase isoenzyme (CK-MB) in mice

[0076] Serum myocardial enzyme analysis showed that the heart of the heat-exposed group was smaller and darker in color compared with the control group; the heart of the preventive probiotic group was larger and lighter in color, closer to the normal heart morphology ( Figure 9 A). Compared with the control group, LDH levels in heat-exposed mice were significantly upregulated; compared with the heat-exposed group, preventive probiotic intervention could downregulate LDH levels ( Figure 9 B). Compared with the control group, the CK-MB level in heat-exposed mice was increased; compared with the heat-exposed group, the preventive probiotic intervention could downregulate the CK-MB level ( Figure 9 C). LDH is an important cellular metabolic enzyme that is highly sensitive to tissue damage and can serve as a diagnostic marker in the late stages of acute myocardial infarction. CK-MB is a sensitive marker of myocardial tissue damage. When myocardial tissue damage is severe, CK-MB is released into the bloodstream, and serum CK-MB levels become a key criterion for diagnosing acute myocardial infarction. These results demonstrate that preventive probiotic intervention significantly alleviates cardiac morphology and myocardial damage in heat-exposed mice.

[0077] Example 11: Effects of the Six-linked Probiotic Preparation on Cardiac Function in Mice

[0078] Compared with the control group, the heart volume level and function level of the heat-exposed mice were significantly decreased, and the heart structure was significantly changed; compared with the heat-exposed group, preventive probiotic intervention significantly improved the heart structure and function of the heat-exposed mice, increased the volume level ( Figure 10 A).

[0079] The above experimental results show that compared with the control group, the left ventricular end-diastolic septal thickness (IVSd), left ventricular posterior wall thickness (LVPWD), and left ventricular free wall thickness (LVPWs) of the heat-exposed group mice were significantly increased, the left ventricular end-diastolic internal diameter (LVIDd) and left ventricular end-systolic internal diameter (LVIDs) were significantly decreased, and the left ventricular end-systolic septal thickness (IVSs) did not change significantly; compared with the heat-exposed group, the IVSd of the probiotic intervention group mice was significantly decreased ( Figure 10 B) LVIDd has an increasing trend ( Figure 10 C) LVPWD decreased significantly ( Figure 10 D) There was no significant change in IVSs ( Figure 10 E), LVIDs increased significantly ( Figure 10 F), LVPWs decreased significantly ( Figure 10G). Among structural indices, IVSd, LVIDd, LVPWD, IVSs, LVIDs, and LVPWs are important indicators for assessing cardiac structure. Increased levels of IVSd and IVSs indicate increased ventricular septum thickness and are important indicators for assessing myocardial hypertrophy. Decreased levels of LVIDd and LVIDs indicate a smaller left ventricle and serve as hallmark indicators of cardiac shrinkage. LVPWD and LVPWs represent the thickness of the left ventricular posterior wall and free wall, respectively, and can assist in assessing the condition of the myocardium. Exposure to extreme heat can lead to cardiac shrinkage and hypertrophy; preventive probiotic intervention can improve cardiac structure in mice exposed to extreme heat.

[0080] Among the functional indicators, compared with the control group, the functional indicators of mice in the heat exposure group, including stroke volume (SV), left ventricular short axis shortening (FS), ventricular septum systolic thickening (IVS), cardiac output (CO), ejection fraction (EF), and left ventricular posterior wall systolic thickening (LVPW), were significantly decreased; compared with the heat exposure group, SV ( Figure 10 H), FS( Figure 10 I), IVS( Figure 10 J), CO( Figure 10 K)、EF( Figure 10 L), and LVPW( Figure 10 M) increased significantly. SV refers to the amount of blood ejected in one heartbeat. The levels of FS and EF are important indicators for evaluating the systolic function of the left ventricle and reflect the myocardial contractility. CO is the cardiac output per minute, and its level can measure the heart's pumping function. The levels of IVS and LVPW reflect the degree of thickening of the ventricular septum and the posterior wall of the left ventricle during systole, reflecting the contractile performance of the myocardium. When the structural level of the heart changes, it first enters the compensatory stage, with a decrease in inner diameter and thickening of the myocardium. When damage continues to occur, it enters the decompensatory stage, with a decrease in volume and a decline in function, and eventually develops into heart failure. The decrease in the above functional indicators in heat-exposed mice indicates that the heart function of heat-exposed mice is impaired. Preventive probiotic intervention can increase the levels of these indicators and improve the heart function of mice after extreme high temperature exposure.

[0081] In terms of volumetric indicators, compared with the control group, the left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) of heat-exposed mice were significantly decreased; compared with the heat-exposed group, the LVEDV ( Figure 10 N) and LVESV( Figure 10O) significantly increased. Among volumetric indices, LVEDV and LVESV represent the volume of the left ventricle at end-diastole and end-systole, reflecting cardiac preload and pumping function. Heat-exposed mice showed a significant decrease in these volumetric indices. Preventive probiotic intervention increased these levels and improved cardiac volume in mice exposed to extreme heat. In summary, preventive probiotic intervention can improve cardiac structure and function in mice exposed to extreme heat.

[0082] Example 12: Effects of the Six-Combination Probiotic Preparation on Cardiac Inflammation in Mice

[0083] Compared with the control group, interleukin-6 (IL-6) was significantly upregulated in the extreme high temperature exposure group; compared with the heat exposure group, probiotic intervention could significantly downregulate the level of IL-6 ( Figure 11 IL-6 is an inflammatory factor involved in the pathogenesis of various infectious and inflammatory diseases. Elevated levels of IL-6 indicate increased inflammation in the body. Preventive probiotic intervention can downregulate IL-6 levels and significantly improve cardiac inflammation in heat-exposed mice.

[0084] Example 13: Effects of the six-combination probiotic preparation on cardiac oxidative stress in mice Compared with the control group, the level of peroxidase-1 (HO-1) was significantly upregulated in the extreme high temperature exposure group; compared with the heat exposure group, the probiotic intervention could significantly downregulate the level of HO-1 ( Figure 12 HO-1 is an important antioxidant enzyme. HO-1 mRNA and protein expression can be upregulated following oxidative stress and cellular damage, and HO-1 is also thought to be involved in vascular protection. HO-1 levels in cardiac tissue are significantly elevated following heat exposure, and preventive probiotic intervention can alleviate heat-induced redox imbalance and restore antioxidant enzyme levels.

Claims

1. A six-linked probiotic for preventing severe heatstroke, characterized in that: The six-combination probiotics include: Bacillus coagulans XY2, Lactobacillus plantarum KL100, Lactobacillus rhamnosus TR08, Lactobacillus helveticus TR05, Lactobacillus casei TR01 and Bifidobacterium longum TR17, and the probiotics are mixed in a ratio of 1:1:1:1:1:1:1 in terms of the number of live bacteria.

2. Use of the six-linked probiotics according to claim 1 in preparing medicines for preventing and treating severe heatstroke.

3. Use of the six-linked probiotics according to claim 1 in the preparation of a drug for enhancing the intestinal mucosal barrier function caused by severe heatstroke.

4. Use of the six-linked probiotics according to claim 1 in the preparation of a drug for preventing severe heatstroke combined with liver damage.

5. The use according to claim 4, characterized in that The heat stroke combined with liver damage is heat stroke combined with acute liver damage.

6. Use of the six-linked probiotics according to claim 1 in the preparation of a drug for preventing severe heatstroke combined with lung injury.

7. The use according to claim 6, characterized in that The heat stroke combined with lung injury is heat stroke combined with acute lung injury.

8. Use of the six-linked probiotics according to claim 1 in the preparation of a drug for preventing severe heatstroke combined with myocardial injury.

9. The use according to claim 8, characterized in that The heat stroke combined with myocardial injury is heat stroke combined with acute myocardial injury.

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