Construction and evaluation method of heat stroke myocardial injury mouse model

Abstract

The present application relates to the technical field of pathological evaluation, and particularly relates to a heat stroke myocardial injury mouse model construction and evaluation method, comprising constructing a heat stroke myocardial injury mouse model by increasing the core body temperature, determining the key time point of the intervention effect of different dose gradients of GDF15 after the heat stroke modeling; obtaining the cTn-I index and the ALT index based on the key time point, determining the injury type ratio based on the normalized values of the cTn-I index and the ALT index to determine the injury type of the heat stroke; adjusting the evaluation strategy in response to the injury type, obtaining the recovery rate of the cTn-I index and the ALT index of the model control group at each time point to determine the myocardial injury degree and the liver injury degree at the corresponding time point; obtaining the average recovery rate of the ALT of each dose group to determine the ALT recovery rate ratio between different dose groups, and adjusting the observation length of the dose group. The present application can distinguish the injury types by constructing the heat stroke myocardial injury model, and cannot adapt to the dynamic changes of different injury types.

Description

Technical Field

[0001] This invention relates to the field of pathological evaluation technology, and in particular to a method for constructing and evaluating a mouse model of myocardial injury caused by heatstroke. Background Technology

[0002] Heatstroke (HS) is a serious and fatal disease with a sudden onset and rapid progression. HS is characterized by a core body temperature >40°C and central nervous system dysfunction, accompanied by damage to multiple organs. Based on the differences in causative factors and high-risk populations, heatstroke is divided into exertional heatstroke (EHS) and classic heatstroke (CHS). The incidence of myocardial injury in heat-related diseases can reach 43.4%-65.2%. The heart is the first key target organ affected by HS. In clinically recovered HS patients, myocardial inflammation and edema can persist for more than 3 months, increasing the long-term risk of cardiovascular events by 1.8-3.9 times. Currently, HS treatment focuses on early cooling, controlling systemic inflammatory response, and multi-organ function support; there is a lack of specific clinical prevention and treatment methods for myocardial injury caused by heatstroke.

[0003] Previous studies have shown that growth differentiation factor-15 (GDF15) can protect against myocardial injury induced by acute stress events and reduce cell damage caused by hypoxia and oxidative stress. Hyperthyroidism (HS) is accompanied by severe oxidative stress, but the role of GDF15 in this pathological process remains unclear. Further research is needed to explore the protective effects and molecular mechanisms of GDF15 in HS-related myocardial injury, providing a new target for the protection of myocardial injury in clinical HS.

[0004] Chinese Patent Publication No. CN120323405A discloses a method for constructing a myocardial ischemia-reperfusion injury model. A vascular occlusion device is inserted into a blood vessel of a target mouse and delivered to the location of the coronary artery where occlusion is to be formed. The vascular occlusion device is used to block the location of the coronary artery where occlusion is to be formed. The vascular occlusion device is partially or entirely made of biodegradable material. The degradation and perforation of the vascular occlusion device within the target mouse body simulates myocardial ischemia-reperfusion injury. Therefore, the method for constructing the myocardial ischemia-reperfusion injury model has the following problems: Existing animal models of heatstroke mainly focus on the model construction itself, lacking a systematic and dynamic evaluation method for myocardial injury after model construction. In particular, after the model is successfully determined, there is a lack of a comprehensive evaluation system for the temporal changes of myocardial injury markers, the dynamic evolution of cardiac function, and the pathological characteristics of myocardial tissue. This makes it difficult to accurately reflect the occurrence, development, and recovery patterns of myocardial injury caused by heatstroke, thus restricting the accurate evaluation of relevant protective strategies. Existing evaluation methods for heatstroke myocardial injury models cannot distinguish between "simple myocardial injury" and "mixed multi-organ injury," leading to inappropriate selection of evaluation indicators; fixed evaluation time points cannot adapt to the dynamic changes of different injury types; and single indicators will be distorted when the recovery of multiple organs is asynchronous. Summary of the Invention

[0005] Therefore, this invention provides a method for constructing and evaluating a mouse model of heatstroke myocardial injury, in order to overcome the problems in the prior art that it cannot distinguish the type of injury, cannot adapt to the dynamic changes of different types of injury, and that the organ recovery is asynchronous and the single indicator is distorted.

[0006] To achieve the above objectives, this invention provides a method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury, comprising: A mouse model of heatstroke-induced myocardial injury was established by raising core body temperature, and the key time points of the intervention effect of different dose gradients of GDF15 after heatstroke modeling were determined. Based on the key time points, the cTn-I index and ALT index are obtained, and the damage type ratio is determined based on the normalized values ​​of the cTn-I index and ALT index to determine the damage type of heatstroke. In response to the injury type regulation and evaluation strategy, the recovery rates of cTn-I and ALT indices at each time point in the model control group were obtained to determine the degree of myocardial injury and liver injury at the corresponding time points. The peak times of cTn-I and ALT indices in the model control group and intervention group were obtained, and the rate of decline of cTn-I and ALT indices in each group was determined to determine the recovery rate ratio between the model control group and the intervention group, and to determine whether the GDF15 injury intervention met expectations. Based on the recovery rate ratio of each group, the peak damage ratio of each indicator is obtained to determine the protective effect index of GDF15 on myocardial and liver function. Based on the protective effect index, the GDF15 damage intervention is verified to see if it meets the expectations and a comprehensive protective index adjustment and evaluation strategy is determined. The average ALT recovery rate of each dose group was obtained based on the ALT recovery rate at each time point of each dose gradient, so as to determine the ratio of ALT recovery rates between different dose groups and adjust the observation time of the dose groups.

[0007] Furthermore, the criteria for successful establishment of a classic heatstroke model is a core body temperature of 42.7°C, and the critical time point for the GDF15 intervention effect is three hours after successful model establishment.

[0008] Furthermore, serum cTn-I and ALT levels were obtained at key time points, and the ratio of the injury type was determined by combining the mean cTn-I and ALT levels of the normal control group. If the injury type ratio is greater than or equal to the ratio threshold, then myocardial injury is judged to be significantly more severe than liver injury, and the injury type of classic heatstroke is myocardial injury-dominant. If the injury type ratio is less than the ratio threshold, it is determined that the degree of myocardial injury and liver injury are similar, and the injury type of classic heatstroke is a mixed injury type.

[0009] Furthermore, at five time points after successful modeling, the cTn-I and ALT indices of the model control group were obtained to determine the cTn-I recovery rate and ALT recovery rate at each time point. The cTn-I recovery rate is the proportion of the current degree of myocardial injury at the corresponding time point relative to the most severe moment, and the ALT recovery rate is the proportion of the current degree of liver injury at the corresponding time point relative to the most severe moment. The recovery rate is determined based on the cTn-I recovery rate and the ALT recovery rate.

[0010] Furthermore, the type of heatstroke injury was determined to be mixed, and the recovery rate ratio between the model control group and the intervention group was determined based on the rate of decline of each indicator. If the recovery rate ratio of the intervention group is greater than that of the model control group, it is determined that GDF15 accelerates myocardial recovery and has better protection for the heart than for the liver. If the recovery rate ratio of the intervention group is less than that of the model control group, it is determined that GDF15 slows down myocardial recovery and has better protection for the liver than for the heart.

[0011] Furthermore, GDF15 showed better protection for the heart than for the liver, confirming that GDF15 intervention can effectively reduce heatstroke-induced myocardial damage in mice, as expected. GDF15 showed better protection for the liver than the heart. The peak injury ratio was determined based on the recovery rate ratio of the intervention group and the recovery rate ratio of the model control group, and the protective index of GDF15 on the myocardium and liver was verified.

[0012] Furthermore, a comprehensive protection index was determined based on the protective effect indices of myocardial function and liver function, and the comprehensive protection index was the average of the protective effect indices of myocardial function and liver function.

[0013] Furthermore, if the protective effect index of myocardial function is greater than that of liver function, it is determined that the evaluation index of GDF15’s protection of the liver is superior to that of the heart, and the evaluation index is output. If the protective effect index of myocardial function is less than that of liver function, it is judged that the protective effect index does not meet expectations.

[0014] Furthermore, the degree of protection of GDF15 against heatstroke injury was determined based on the comprehensive protection index; If the comprehensive protection index is greater than or equal to the first index threshold, then GDF15 is judged to have strong protection against heatstroke damage, and the dose of GDF15 intervention can be reduced to conduct a dose-response optimization experiment. If the comprehensive protection index is less than the first index threshold but greater than or equal to the second index threshold, then GDF15 is judged to provide moderate protection against heatstroke injury, and the observation time needs to be extended to confirm the integrity of recovery. If the overall protection index is less than the second index threshold, then GDF15 is judged to provide weak protection against heatstroke damage, and the GDF15 intervention dose is increased for re-evaluation.

[0015] Furthermore, the ratio of ALT recovery rate between different dose groups includes the ratio of ALT recovery rate of the medium dose group to that of the high dose group, denoted as the medium-high recovery ratio, and the ratio of ALT recovery rate of the low dose group to that of the high dose group, denoted as the low-high recovery ratio. If the ratio of medium-high recovery is less than or equal to the first recovery threshold, and the ratio of low-high recovery is less than or equal to the second recovery threshold, then it is determined that there is differential recovery in GDF15 dosage, and the observation period of the low-dose group is extended.

[0016] Compared with existing technologies, the beneficial effects of this invention are as follows: This invention, through multi-organ biochemical indicators, cardiac function, and pathological dynamic monitoring at multiple time points, determines that 3 hours after successful modeling is the critical time window for synchronous abnormalities in cardiac, liver, and kidney function, providing a precise time point for evaluating the GDF15 intervention effect; secondly, by setting up a parallel comparison between a PBS solvent control and high, medium, and low dose GDF15 intervention groups, combined with non-targeted lipid metabolomics and the detection of key ferroptosis protein expression, a multi-level evaluation system from myocardial injury markers and cardiac function to molecular mechanisms is established; this method achieves thermal radiation through the normalized ratio of cTn-I to ALT. Precise branching of myocardial injury types avoids misjudgment based on a single indicator; a quantitative evaluation system for the organ protective effect of GDF15 is constructed based on the recovery rate ratio and peak injury ratio, distinguishing the differential protection of GDF15 on the heart and liver; the observation time of different dose groups is adaptively adjusted by the ALT recovery rate ratio R_ALT, so that the evaluation time point matches the actual recovery speed of each dose group, effectively avoiding false negative evaluation errors caused by differences in recovery rate; this method realizes a complete closed loop from injury classification, quantification of protective effect to adaptive evaluation scheme, significantly improving the accuracy and repeatability of the evaluation of heatstroke myocardial injury model.

[0017] Furthermore, among the myocardial injury-related biomarkers, cTn-I is a specific indicator of myocardial cell injury, and ALT is a liver function-related indicator of myocardial cell injury. This invention is based on the normalized ratio of cTn-I to ALT to determine the type of heatstroke injury. By detecting serum cTn-I and ALT at the critical time point of 3 hours after successful modeling and calculating the injury type ratio, an objective quantitative branch judgment of the type of myocardial injury in heatstroke is achieved. The myocardial injury-dominant type can trigger a simplified evaluation process, and myocardial injury can be effectively assessed by detecting only cTn-I, EF, and myocardial pathology, which significantly shortens the evaluation cycle and reduces the detection cost. The mixed injury type indicates that a complete set of heart, liver, and kidney tests should be retained to avoid evaluation bias caused by a single myocardial indicator. The method establishes a clear quantitative threshold based on experimental data, which effectively solves the technical problem of inconsistent evaluation standards for multi-organ injury in heatstroke models and improves the accuracy and efficiency of evaluation.

[0018] Furthermore, this invention introduces the recovery rate ratio and peak injury ratio to quantitatively distinguish the differential protective effects of GDF15 on the heart and liver. Liver protection is superior to cardiac protection. By calculating the protective effect indices of the myocardium and liver, a special protective mode with strong acute protective effects but weak recovery ability is identified, avoiding misjudgments that may be caused by a single recovery rate ratio indicator. Based on the comparison of the comprehensive protection index and the threshold, the evaluation strategy is adaptively adjusted: the observation time is extended for moderate protection, the dose is reduced for strong protection, and the dose is reassessed for weak protection. This method effectively solves the technical problem of inconsistent evaluation standards for the organ protective effects of GDF15.

[0019] Furthermore, when the protective effect index does not meet expectations, this invention establishes an adaptive observation duration adjustment mechanism based on the difference in recovery rate by calculating the average ALT recovery rate and its ratio (medium-high recovery ratio, low-high recovery ratio) of different dose GDF15 intervention groups. When there is a dose difference recovery, the observation duration of the low-dose group is extended. The adjustment mechanism effectively avoids false negative evaluation errors caused by the difference in recovery rate between different dose groups, and makes the evaluation time point of each dose group match its actual recovery speed, which significantly improves the accuracy and fairness of the evaluation results in multi-dose comparison experiments. Attached Figure Description

[0020] Figure 1 This is a flowchart illustrating the construction and evaluation method of a mouse model of heatstroke myocardial injury in an embodiment of the present invention. Figure 2 This is the core body temperature change curve of a heatstroke model mouse during heat exposure in an embodiment of the present invention; Figure 3 The highest core body temperature and modeling time during modeling in each group of embodiments of the present invention; Figure 4This invention illustrates the effect of GDF15 intervention on core body temperature changes in heatstroke mice during modeling in each group of the embodiments of the present invention. Figure 5 This is a comparison of modeling time and peak core body temperature at onset among different intervention groups in this embodiment of the invention. Detailed Implementation

[0021] To make the objectives and advantages of the present invention clearer, the present invention will be further described below with reference to embodiments; it should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0022] Preferred embodiments of the present invention will now be described with reference to the accompanying drawings. Those skilled in the art should understand that these embodiments are merely illustrative of the technical principles of the present invention and are not intended to limit the scope of protection of the present invention.

[0023] It should be noted that in the description of this invention, the terms "upper", "lower", "left", "right", "inner", "outer", etc., which indicate directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings. This is only for the convenience of description and is not intended to indicate or imply that the device or element must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, it should not be construed as a limitation of this invention.

[0024] Furthermore, it should be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0025] Please see Figures 1-5 As shown, Figure 1 This is a flowchart illustrating the construction and evaluation method of a mouse model of heatstroke myocardial injury in an embodiment of the present invention. Figure 2 This is the core body temperature change curve of a heatstroke model mouse during heat exposure in an embodiment of the present invention; Figure 3 The highest core body temperature and modeling time during modeling in each group of embodiments of the present invention; Figure 4 This invention illustrates the effect of GDF15 intervention on core body temperature changes in heatstroke mice during modeling in each group of the embodiments of the present invention. Figure 5 This is a comparison of modeling time and peak core body temperature at onset among different intervention groups in this embodiment of the invention.

[0026] This invention provides a method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury, comprising: Step S1: A mouse model of heatstroke myocardial injury was constructed by raising core body temperature, and the key time points of the intervention effect of different dose gradients of GDF15 after heatstroke modeling were determined. Step S2: Obtain the cTn-I index and ALT index based on the key time points, and determine the damage type ratio based on the normalized values ​​of the cTn-I index and ALT index to determine the damage type of heatstroke. Step S3: In response to the injury type adjustment evaluation strategy, obtain the recovery rates of cTn-I and ALT indices at each time point in the model control group to determine the degree of myocardial injury and liver injury at the corresponding time points. Step S4: Obtain the peak times of cTn-I and ALT indices in the model control group and intervention group, determine the rate of decrease of cTn-I and ALT indices in each group, and determine the recovery rate ratio between the model control group and the intervention group to determine whether the GDF15 injury intervention meets expectations. Step S5: Based on the recovery rate ratio of each group, obtain the peak damage ratio of each indicator to determine the protective effect index of GDF15 on myocardial and liver function. Based on the protective effect index, verify whether the GDF15 damage intervention meets expectations and determine the comprehensive protection index adjustment and evaluation strategy. Step S6: Based on the ALT recovery rate of each dose gradient at each time point, obtain the average ALT recovery rate of each dose group to determine the ratio of ALT recovery rates between different dose groups and adjust the observation time of the dose groups.

[0027] Specifically, this invention achieves accurate branching of heatstroke myocardial injury types by using the cTn-I to ALT normalized ratio, avoiding misjudgment based on a single indicator; it constructs a quantitative evaluation system for the organ protective effect of GDF15 based on the recovery rate ratio and the peak injury ratio, distinguishing the differential protection of GDF15 on the heart and liver; it adaptively adjusts the observation time of different dose groups by using the ALT recovery rate ratio R_ALT, matching the evaluation time point with the actual recovery speed of each dose group, effectively avoiding false negative evaluation errors caused by differences in recovery rate; this method realizes a complete closed loop from injury classification, quantification of protective effect to adaptive evaluation scheme, significantly improving the accuracy and repeatability of heatstroke myocardial injury model evaluation.

[0028] In this embodiment, the heatstroke (HS) type is classic heatstroke (CHS).

[0029] Methods for constructing a mouse model of heatstroke-induced myocardial injury include: A CHS mouse model was constructed using a constant temperature and humidity chamber (temperature 40°C, relative humidity 60%). An implantable telemetry technology was used to monitor the core body temperature of the mice in real time by implanting a temperature-measuring capsule in the peritoneum. A core body temperature of 42.7°C was used as the criterion for successful CHS modeling.

[0030] Specifically, mice were randomly assigned to groups of 6 mice each. On the day of the experiment, all mice were fasted and deprived of water. To avoid the influence of circadian rhythms, the experiment was scheduled to begin between 9:00 and 10:00 AM daily. Before the experiment, the constant temperature and humidity chamber was preheated to 40±1°C and 60±5% relative humidity. The core body temperature of the mice was recorded one hour before modeling. Once the body temperature stabilized below 37.5°C, the mice were moved into the constant temperature and humidity chamber. Heatstroke was diagnosed by real-time monitoring of core body temperature. In this study, the criterion for heatstroke was set as a core body temperature ≥42.7°C [22,34]. Considering the real-time fluctuations in the core body temperature of mice during the experiment, the time corresponding to three consecutive temperature measurements ≥42.7°C was considered the successful modeling point. During modeling, the control group mice were placed in a room temperature environment. After successful modeling, the mice were immediately removed from the constant temperature and humidity chamber and placed in a room temperature environment.

[0031] Five different time points were set after successful CHS modeling: HS-0h group (immediately after successful modeling), HS-3h group, HS-6h group, HS-12h group and HS-24h group, and normal control group (Ctrl group).

[0032] The dynamic changes of cardiac troponin I (cTn-I), creatine kinase isoenzyme MB (CK-MB), and lactate dehydrogenase (LDH) markers of myocardial injury were detected in CHS mice.

[0033] During the experiment, a fully automated biochemical analyzer was used to measure serum liver function indicators alanine aminotransferase (ALT) and aspartate aminotransferase (AST), kidney function indicators creatinine (CREA) and blood urea nitrogen (BU), and myocardial injury indicator lactate dehydrogenase (LDH) activity. Enzyme-linked immunosorbent assay (ELISA) was used to measure the concentrations of cardiac troponin I (cTn-I) and creatine kinase isoenzyme MB (CK-MB).

[0034] In addition, continuous dynamic echocardiography was performed on CHS mice at the above 5 time points. Combined with changes in myocardial injury markers and cardiac function indicators, and supplemented by myocardial tissue pathological examination, the key time points for subsequent observation of the GDF15 intervention effect were determined.

[0035] In this embodiment, there were no significant differences in the time of successful modeling and the highest core body temperature at the onset of disease among the different modeling groups. Three hours after successful modeling (HS-3h) was the critical time point for myocardial enzymes and liver function indicators to simultaneously reach their peak values, while kidney function indicators remained abnormal. Echocardiography results showed that myocardial contractility significantly decreased after HS, with persistent impairment within 24 hours, and significant cardiac function suppression in HS-3h mice. Pathological observation of myocardial tissue at HS-3h revealed myocardial cell edema, disordered arrangement, and significant inflammatory cell infiltration. Based on the comprehensive analysis of core body temperature, biochemical indicators, cardiac function, and myocardial pathological changes, 3 hours after successful HS modeling was ultimately determined as the time point for observing the GDF15 intervention effect, as this time point can comprehensively reflect the multi-organ damage state induced by HS.

[0036] Three dose gradients of GDF15 were set up: high (30 μg / kg), medium (6 μg / kg), and low (1.2 μg / kg). Mice were injected subcutaneously 1 hour before modeling.

[0037] Six experimental groups were set up: normal control group (Ctrl), Ctrl + high-dose GDF15 group (Ctrl + G), HS + PBS group (HS), HS + low-dose GDF15 group (HS + GL), HS + medium-dose GDF15 group (HS + GM), and HS + high-dose GDF15 group (HS + GH).

[0038] Serum myocardial enzymes, liver and kidney function indicators were measured in mice of each group. Cardiac function was examined and myocardial tissue pathological changes were observed to comprehensively evaluate the protective effect of GDF15 against CHS myocardial injury.

[0039] The HS+PBS group (HS) refers to the control group of the heatstroke model. Specifically: The mice in this group also underwent heatstroke (CHS) modeling treatment, but were injected subcutaneously with an equal amount of PBS (phosphate-buffered saline) one hour before modeling, instead of being injected with GDF15 protein.

[0040] PBS was used as a solvent control for GDF15 to eliminate the influence of injection procedures and the solvent itself on the experimental results, ensuring that the observed effects did indeed come from the intervention of GDF15.

[0041] Therefore, the PBS group served as both a negative control and a solvent control, and is an important benchmark for evaluating the effectiveness of GDF15 intervention.

[0042] Non-targeted lipid metabolomics was performed on myocardial tissues from HS and Ctrl mice to analyze the lipid metabolism profile of CHS mice myocardium. Western blotting (WB) was used to detect the expression of key proteins related to lipid metabolism and ferroptosis, exploring the regulatory effect of GDF15 on the core ferroptosis pathway.

[0043] Specifically, this invention, through multi-organ biochemical indicators, cardiac function, and pathological dynamic monitoring at multiple time points, identifies 3 hours after successful modeling as the critical time window for synchronous abnormalities in cardiac, hepatic, and renal functions, providing a precise time point for evaluating the GDF15 intervention effect. Secondly, by setting up a parallel comparison between a PBS solvent control and high, medium, and low dose GDF15 intervention groups, combined with non-targeted lipid metabolomics and the detection of key ferroptosis protein expression, a multi-level evaluation system from myocardial injury markers and cardiac function to molecular mechanisms was established. Three hours after successful modeling, serum cTn-I and ALT levels were measured. The unit for cTn-I was pg / ml, and the unit for ALT was U / L. The damage type ratio was calculated based on the normalized values ​​of cTn-I and ALT levels. Specifically, the injury type ratio = (cTn-I index / mean cTn-I index in the normal control group) / (ALT index / mean ALT index in the normal control group); If the injury type ratio is greater than or equal to the ratio threshold, then myocardial injury is judged to be significantly more severe than liver injury, and the injury type of classic heatstroke is myocardial injury-dominant. If the injury type ratio is less than the ratio threshold, it is determined that the degree of myocardial injury and liver injury are similar, and the injury type of classic heatstroke is a mixed injury type.

[0044] The ratio threshold is 1.5.

[0045] Heatstroke injury is predominantly myocardial, and subsequent evaluation can simplify liver and kidney function tests and focus on myocardial specific indicators. The type of damage from heatstroke is a mixed type. Subsequent evaluation should retain a full panel of tests on the heart, liver, and kidneys; otherwise, the multi-organ interaction effects may be underestimated. Understandably, the simplified evaluation process triggered by myocardial injury only detects cTn-I and EF, as well as myocardial pathology to shorten the evaluation cycle. In the mixed injury type, a single myocardial indicator cannot accurately reflect the model status.

[0046] In this embodiment, among the myocardial injury-related biomarkers, cTn-I is a specific indicator of myocardial cell injury. Its serum level increased significantly immediately upon successful HS-0h model establishment, and the difference compared with the control group was statistically significant. The cTn-I index was 56.43±6.21 vs 36.88±3.00 pg / mL compared with the control group (P<0.01). The serum level gradually increased with the extension of recovery time, reaching a peak at 6h, where the cTn-I index was 84.12±13.30 vs 36.88±3.00 pg / mL compared with the control group (P<0.001). Although it showed a decreasing trend afterward, the level at 24h was still significantly higher than that of the blank control group, where the cTn-I index was 64.01±8.92 vs 36.88±3.00 pg / mL compared with the control group (P<0.001).

[0047] The liver function-related indicators ALT and AST showed similar trends, while both indicators increased in HS-0h. During the implementation, the liver function-related indicators ALT and AST in HS-0h were 86.95±21.87 vs 43.10±16.05 U / L, P<0.001, and 211.73±17.05 vs 107.50±14.15 U / L, P<0.001. Liver function related indicators ALT and AST reached their peak at 3 hours. During the procedure, ALT was 119.31±16.78 vs 43.10±16.05 U / L (P<0.001), and AST was 271.35±44.29 vs 107.50±14.15 U / L (P<0.001), and then slowly decreased.

[0048] ALT levels returned to normal at 24 hours, showing no significant difference from the control group. The ALT level during the study was 59.00±8.07 vs 43.10±16.05 U / L (P>0.05). However, AST levels at 24 hours were still slightly higher than the control group, at 168.40±42.54 vs 107.50±14.15 U / L (P<0.05).

[0049] Specifically, among the myocardial injury-related biomarkers, cTn-I is a specific indicator of myocardial cell injury, and ALT is a liver function-related indicator of myocardial cell injury. This invention is based on the normalized ratio of cTn-I to ALT to determine the type of heatstroke injury. By detecting serum cTn-I and ALT at the critical time point of 3 hours after successful modeling and calculating the injury type ratio, an objective quantitative branch judgment of the myocardial injury type in heatstroke is achieved. The myocardial injury-dominant type can trigger a simplified evaluation process, and myocardial injury can be effectively assessed by detecting only cTn-I, EF, and myocardial pathology, which significantly shortens the evaluation cycle and reduces the detection cost. The mixed injury type indicates that a complete set of tests for the heart, liver, and kidneys should be retained to avoid evaluation bias caused by a single myocardial indicator. The method establishes a clear quantitative threshold based on experimental data, which effectively solves the technical problem of inconsistent evaluation standards for multi-organ injury in heatstroke models and improves the accuracy and efficiency of evaluation.

[0050] Five time points after successful modeling were observed, and the cTn-I and ALT indices of the model control group were measured. The cTn-I recovery rate and ALT recovery rate at each time point were calculated. cTn-I recovery rate = cTn-I index at each time point / maximum value of cTn-I index. In practice, the peak value of cTn-I index appeared at HS-6h, which was 84.12 pg / mL. It is understandable that the cTn-I recovery rate represents the proportion of the current degree of myocardial damage relative to the most severe moment at the corresponding time point. If HS-24h=0.76, it means that the damage is still maintained at 76% of the peak value after 24 hours, indicating slow recovery.

[0051] ALT recovery rate = ALT index at each time point / maximum value of ALT index. In practice, the peak value of ALT index occurred at HS-3h, which was 119.31U / L. It is understandable that the ALT recovery rate represents the proportion of the current degree of liver damage relative to the most severe moment at the corresponding time point. If HS-12h=0.65, it means that the liver damage has dropped to 65% of the peak value at 12 hours, and the recovery is relatively obvious.

[0052] The heatstroke injury type was a mixed type. The peak times of cTn-I and ALT in the model control group and the GDF15 intervention group were detected. The peak time of cTn-I in the model control group was 6 hours after successful modeling. The rate of decrease of cTn-I index in the model control group = (cTn-I index at peak time - cTn-I index 18 hours after peak time) / 18; The rate of decrease of ALT index in the model control group = (ALT index at peak time - ALT index 18 hours after peak time) / 18; The rate of decrease of cTn-I index in the intervention group = (cTn-I index at peak time - cTn-I index 18 hours after peak time) / 18; The rate of decrease of ALT index in the intervention group = (ALT index at peak time - ALT index 18 hours after peak time) / 18; The recovery rate ratio between the model control group and the intervention group is determined based on the rate of decline of each indicator. The recovery rate ratio of the model control group = the rate of decline of the cTn-I indicator in the model control group / the rate of decline of the ALT indicator in the model control group. The recovery rate ratio of the intervention group = the rate of decline of the cTn-I indicator in the intervention group / the rate of decline of the ALT indicator in the intervention group. If the recovery rate ratio of the intervention group is greater than that of the model control group, it is determined that GDF15 accelerates myocardial recovery and has better protection for the heart than for the liver. If the recovery rate ratio of the intervention group is less than that of the model control group, it is determined that GDF15 slows down myocardial recovery and has better protection for the liver than for the heart. GDF15 showed better protection for the heart than the liver, confirming that GDF15 intervention can effectively reduce heatstroke-induced myocardial damage in mice, as expected. GDF15 showed better protection for the liver than the heart. The peak injury ratio was determined based on the recovery rate ratio of the intervention group and the recovery rate ratio of the model control group, and the protective index of GDF15 on the myocardium and liver was verified.

[0053] In this embodiment, after intervention with different doses of GDF15, the levels of the above-mentioned myocardial injury markers in mice in each HS intervention group decreased to varying degrees, and it was found that the improvement effect of the relevant indicators was correlated with the intervention dose of GDF15.

[0054] Among them, the cTn-I level in the low, medium and high dose GDF15 intervention groups was significantly lower than that in the HS model control group, and the differences were statistically significant. The cTn-I levels in the low, medium and high dose GDF15 intervention groups were 50.12±3.17 vs 59.31±2.71 pg / mL, P<0.001; 43.96±2.83 vs 59.31±2.71 pg / mL, P<0.001; 40.45±3.96 vs 59.31±2.71 pg / mL, P<0.001.

[0055] The peak damage ratio is determined based on the recovery rate ratio of the intervention group and the recovery rate ratio of the model control group. The peak damage ratio of cTn-I in the model control group = cTn-I index at peak time / cTn-I index in the normal control group. The peak damage ratio of ALT in the model control group = ALT index at peak time / ALT index in the normal control group. The peak ratio of cTn-I damage in the intervention group = peak cTn-I index / cTn-I index in the normal control group; the peak ratio of ALT damage in the intervention group = peak ALT index / ALT index in the normal control group. The protective effect index of GDF15 on myocardial and liver function was determined based on the peak injury ratio between the model control group and the intervention group. Specifically, the protective effect index of myocardial function = (cTn-I injury peak ratio in the model control group - cTn-I injury peak ratio in the intervention group) / (cTn-I injury peak ratio in the model control group - 1) × 100%, and the protective effect index of liver function = (ALT injury peak ratio in the model control group - ALT injury peak ratio in the intervention group) / (ALT injury peak ratio in the model control group - 1) × 100%.

[0056] Understandably, the protective effect index represents the degree to which GDF15 reduces damage beyond the normal range. Eliminating the influence of differences in absolute heat exposure time, it only reflects the inhibitory effect of GDF15 on additional damage.

[0057] The comprehensive protection index is determined based on the protective effect indices of myocardial function and liver function, and the comprehensive protection index is the average of the protective effect indices of myocardial function and liver function. If the protective effect index of myocardial function is greater than that of liver function, it is determined that the evaluation index of GDF15’s protection of the liver is superior to that of the heart, and the evaluation index is output. If the protective effect index of myocardial function is less than the protective effect index of liver function, it is judged that the protective effect index does not meet expectations. Specifically, the recovery rate ratio reflects the relative rate of recovery, rather than the absolute degree of protection against peak damage. GDF15 reduces peak myocardial damage, but the rate of decrease in myocardial damage during the recovery period is slower than that of the liver, indicating slower myocardial damage recovery. The output evaluation index shows that GDF15 has a strong acute protective effect on the myocardium, but the myocardium's recovery ability is still weaker than that of the liver.

[0058] The degree of protection of GDF15 against heatstroke injury was determined based on the comprehensive protection index, and the evaluation strategy was adjusted accordingly. Specifically, if the comprehensive protection index is greater than or equal to the first index threshold, then GDF15 is judged to have strong protection against heatstroke damage, and the GDF15 intervention dose can be reduced to conduct a dose-response optimization experiment. If the comprehensive protection index is less than the first index threshold but greater than or equal to the second index threshold, then GDF15 is judged to provide moderate protection against heatstroke injury, and the observation time needs to be extended to confirm the integrity of recovery. If the overall protection index is less than the second index threshold, then GDF15 is judged to provide weak protection against heatstroke, and the GDF15 intervention dose is increased for re-evaluation. In this embodiment, the protective effect index of myocardial function is greater than that of liver function, the first index threshold is 70%, and the second index threshold is 40%.

[0059] Specifically, this invention introduces the recovery rate ratio and peak injury ratio to quantitatively distinguish the differential protective effects of GDF15 on the heart and liver. Liver protection is superior to cardiac protection. By calculating the protective effect indices for the myocardium and liver, a special protective mode with strong acute protective effects but weak recovery ability is identified, avoiding misjudgments that may be caused by a single recovery rate ratio indicator. Based on the comparison of the comprehensive protection index and threshold, the evaluation strategy is adaptively adjusted: the observation time is extended for moderate protection, the dose is reduced for strong protection, and the dose is reassessed for weak protection. This method effectively solves the technical problem of inconsistent evaluation standards for the organ protective effects of GDF15.

[0060] When the protective effect index does not meet expectations, obtain the ALT recovery rate of the high-dose group, medium-dose group and low-dose group of GDF15 at each time point, and determine the average ALT recovery rate of each dose group within the current observation period. The average ALT recovery rate for each dose group = (ALT recovery rate from 1 to 24 hours) / (24 - the time point at which the corresponding dose group's ALT peak occurred). The ALT recovery rate ratio between different dose groups is determined based on the average ALT recovery rate of each dose group. The ALT recovery rate ratio between different dose groups includes the ratio of the ALT recovery rate of the medium dose group to the ALT recovery rate of the high dose group, which is denoted as the medium-high recovery ratio, and the ratio of the ALT recovery rate of the low dose group to the ALT recovery rate of the high dose group, which is denoted as the low-high recovery ratio. During implementation, if the ratio of medium-high recovery is less than or equal to the first recovery threshold and the ratio of low-high recovery is less than or equal to the second recovery threshold, it is determined that there is differential recovery of GDF15 dose, and the observation period of the low-dose group is extended to 48 hours. If the low-to-high recovery ratio is greater than the first recovery threshold, then the ALT recovery rates of each dose group are considered to be similar. Wherein, the first recovery threshold is 0.8 and the second recovery threshold is 0.6.

[0061] Specifically, when the protective effect index does not meet expectations, this invention establishes an adaptive observation duration adjustment mechanism based on the difference in recovery rate by calculating the average ALT recovery rate and its ratio (medium-high recovery ratio, low-high recovery ratio) of different dose GDF15 intervention groups. When there is a dose difference recovery, the observation duration of the low-dose group is extended. The adjustment mechanism effectively avoids false negative evaluation errors caused by the difference in recovery rate between different dose groups, and makes the evaluation time point of each dose group match its actual recovery speed, which significantly improves the accuracy and fairness of the evaluation results in multi-dose comparison experiments.

[0062] The technical solution of the present invention has been described above with reference to the preferred embodiments shown in the accompanying drawings. However, it will be readily understood by those skilled in the art that the scope of protection of the present invention is obviously not limited to these specific embodiments. Without departing from the principles of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will all fall within the scope of protection of the present invention.

[0063] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury, characterized in that, include: A mouse model of heatstroke-induced myocardial injury was established by raising core body temperature, and the key time points of the intervention effect of different dose gradients of GDF15 after heatstroke modeling were determined. Based on the key time points, the cTn-I index and ALT index are obtained, and the damage type ratio is determined based on the normalized values ​​of the cTn-I index and ALT index to determine the damage type of heatstroke. In response to the injury type regulation evaluation strategy, the recovery rates of cTn-I and ALT indices at each time point in the model control group were obtained to determine the degree of myocardial and liver injury at the corresponding time points. The recovery rate ratio between the model control group and the intervention group was determined based on the rate of decline of cTn-I and ALT in each group, to determine whether the GDF15 injury intervention met expectations. The rate of decline of cTn-I and ALT in each group was determined based on the peak time of cTn-I and ALT in the model control group and the intervention group. Based on the recovery rate ratio of each group, the peak damage ratio of each indicator is obtained to determine the protective effect index of GDF15 on myocardial and liver function. Based on the protective effect index, the GDF15 damage intervention is verified to see if it meets the expectations and a comprehensive protection index adjustment and evaluation strategy is determined. The average ALT recovery rate of each dose group was obtained based on the ALT recovery rate at each time point of each dose gradient, so as to determine the ratio of ALT recovery rates between different dose groups and adjust the observation time of the dose groups.

2. The method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury according to claim 1, characterized in that, The criteria for successful modeling of classic heatstroke is a core body temperature of 42.7°C, and the critical time point for the GDF15 intervention effect is three hours after successful modeling.

3. The method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury according to claim 2, characterized in that, Serum cTn-I and ALT levels were obtained at key time points, and the ratio of the injury type was determined by combining the mean cTn-I and ALT levels of the normal control group. If the injury type ratio is greater than or equal to the ratio threshold, then myocardial injury is determined to be significantly more severe than liver injury, and the injury type of classic heatstroke is determined to be myocardial injury-dominant. If the injury type ratio is less than the ratio threshold, it is determined that the degree of myocardial injury and liver injury are similar, and the injury type of classic heatstroke is determined to be mixed injury.

4. The method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury according to claim 3, characterized in that, At five time points after successful modeling, the cTn-I and ALT indices of the model control group were obtained to determine the cTn-I recovery rate and ALT recovery rate at each time point. The cTn-I recovery rate is the proportion of the current degree of myocardial injury at the corresponding time point relative to the most severe moment, and the ALT recovery rate is the proportion of the current degree of liver injury at the corresponding time point relative to the most severe moment. The recovery rate is determined based on the cTn-I recovery rate and the ALT recovery rate.

5. The method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury according to claim 4, characterized in that, The damage type of heatstroke is mixed. The recovery rate ratio between the model control group and the intervention group was determined based on the rate of decline of each indicator. If the recovery rate ratio of the intervention group is greater than that of the model control group, it is determined that GDF15 accelerates myocardial recovery and has better protection for the heart than for the liver. If the recovery rate ratio of the intervention group is less than that of the model control group, it is determined that GDF15 slows down myocardial recovery and has better protection for the liver than for the heart.

6. The method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury according to claim 5, characterized in that, GDF15 showed better protection for the heart than the liver, confirming that GDF15 intervention can effectively reduce heatstroke-induced myocardial damage in mice, as expected. GDF15 showed better protection for the liver than the heart. The peak injury ratio was determined based on the recovery rate ratio of the intervention group and the recovery rate ratio of the model control group, and the protective index of GDF15 on the myocardium and liver was verified.

7. The method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury according to claim 6, characterized in that, The comprehensive protection index is determined based on the protective effect indices of myocardial function and liver function. The comprehensive protection index is the average of the protective effect indices of myocardial function and liver function.

8. The method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury according to claim 7, characterized in that, If the protective effect index of myocardial function is greater than that of liver function, it is determined that the evaluation index of GDF15’s protection of the liver is superior to that of the heart, and the evaluation index is output. If the protective effect index of myocardial function is less than that of liver function, it is judged that the protective effect index does not meet expectations.

9. The method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury according to claim 8, characterized in that, The degree of protection of GDF15 against heatstroke injury was determined based on the comprehensive protection index; If the comprehensive protection index is greater than or equal to the first index threshold, then GDF15 is judged to have strong protection against heatstroke damage, and the dose of GDF15 intervention can be reduced to conduct a dose-response optimization experiment. If the comprehensive protection index is less than the first index threshold but greater than or equal to the second index threshold, then GDF15 is judged to provide moderate protection against heatstroke injury, and the observation time needs to be extended to confirm the integrity of recovery. If the overall protection index is less than the second index threshold, then GDF15 is judged to provide weak protection against heatstroke damage, and the GDF15 intervention dose is increased for re-evaluation.

10. The method for constructing and evaluating a mouse model of heatstroke-induced myocardial injury according to claim 9, characterized in that, The ratio of ALT recovery rate between different dose groups includes the ratio of ALT recovery rate of the medium dose group to that of the high dose group, denoted as the medium-high recovery ratio, and the ratio of ALT recovery rate of the low dose group to that of the high dose group, denoted as the low-high recovery ratio. If the ratio of medium-high recovery is less than or equal to the first recovery threshold, and the ratio of low-high recovery is less than or equal to the second recovery threshold, then it is determined that there is differential recovery in GDF15 dosage, and the observation period of the low-dose group is extended.

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