Use of tert-butyl hydroquinone in the preparation of a preparation for alleviating splenic injury caused by gas explosion

Abstract

The present application relates to the application of tert-butyl hydroquinone in the preparation of a preparation for alleviating the spleen injury caused by gas explosion. The present application found that, compared with the untreated model group, the treatment of tert-butyl hydroquinone can significantly weaken the spleen atrophy and inflammatory hemorrhage caused by gas explosion, indicating that tert-butyl hydroquinone can alleviate the degree of rat spleen injury caused by gas explosion, which provides a new treatment scheme for the spleen injury caused by gas explosion and a new research direction for improving the treatment rate of gas explosion injury. At the same time, it is found that tert-butyl hydroquinone can significantly down-regulate the expression of inflammatory factors IL-6 and injury markers ROS in the spleen of the rat model of gas explosion injury, and significantly up-regulate the expression of anti-inflammatory factors IL-10 and oxidative stress regulatory factors HMOX1 and pathway related genes Nrf2, HO-1, COX2 and GPX4.

Description

Technical Field

[0001] This invention relates to the application of tert-butylhydroquinone in the preparation of agents for mitigating spleen damage caused by gas explosions, and belongs to the field of biomedical technology. Background Technology

[0002] Gas explosions are a common disaster that occurs during coal mining when methane gas accumulates to a certain concentration in the air and encounters an open flame. Gas explosions can cause casualties and enormous economic losses, and even restrict the development of the coal industry.

[0003] Because gas explosions produce blast waves, high-temperature flames, and toxic gases, gas explosion injuries are often complex and multiple injuries. Previous studies have shown that gas explosions can cause damage to multiple organs, including the nervous system, lungs, kidneys, and spleen. The spleen, the largest immune organ in the human body, is particularly vulnerable to injury due to its fragile and soft texture. In recent decades, the treatment of spleen injuries has undergone significant changes, considering the impaired immune function and overwhelming postoperative infections following splenectomy. Therefore, non-surgical treatments are increasingly favored to protect the spleen and its function. In conclusion, early spleen-preserving treatment is crucial for patient prognosis in cases of spleen injury caused by gas explosions. Therefore, understanding the molecular mechanisms of spleen injury in its early stages is a key research direction for finding early treatment methods to preserve the spleen.

[0004] Oxidative stress is a stress response in which the body produces reactive oxygen species (ROS) and reactive nitrogen species (RNS) free radicals when exposed to harmful stimuli from the internal and external environment. Oxidative stress occurs when ROS accumulates in cells, and this imbalance severely damages proteins, lipids, and DNA. Previous research on oxidative stress was primarily "superoxide-centric," focusing on the pathological origins of these oxygen-derived free radicals, the types of molecular damage they may cause, and the protection provided by antioxidant enzymes. In recent years, oxidative stress has been viewed as a pathological response caused by an imbalance in genes or gene expression regulation. At the heart of this new focus is nuclear factor E2-related factor 2 (Nrf2). Nrf2 is considered a "master regulator" of antioxidant responses, regulating the expression of hundreds of genes. It activates ARE-dependent gene expression by binding to antioxidant responsive elements (AREs) to form the Nrf2 / ARE complex, exerting its physiological functions of anti-inflammation, anti-oxidation, detoxification, autophagy, and proteasome activity. However, there are no reports on whether the Nrf2 / ARE signaling pathway plays an antioxidant protective role in spleen tissue damage caused by gas explosion. Summary of the Invention

[0005] The purpose of this invention is to provide the application of tert-butylhydroquinone in the preparation of agents for mitigating spleen damage caused by gas explosions, in order to solve the problem that the role of oxidative stress in spleen tissue damage caused by gas explosions is unknown in the prior art.

[0006] To achieve the above objectives, the technical solution for the application of tert-butylhydroquinone in the preparation of agents for mitigating spleen damage caused by gas explosions in this invention is as follows:

[0007] Application of tert-butylhydroquinone in the preparation of agents to mitigate spleen damage caused by gas explosions.

[0008] The beneficial effects of the above technical solution are as follows: The inventors' team previously established a realistic tunnel environment and successfully created an animal model of gas explosion injury, laying the foundation for research on spleen injury caused by gas explosions. The study found that gas explosions lead to severe spleen atrophy in rats, resulting in inflammatory hemorrhage, decreased lymphocytes, and diffuse necrosis; while tert-butylhydroquinone (TBHQ) can alleviate spleen atrophy caused by gas explosions, reducing congestion and hemorrhage in the red pulp of the spleen, indicating that tert-butylhydroquinone can alleviate the degree of spleen injury in rats caused by gas explosions.

[0009] As a further improvement, the method of mitigating spleen damage caused by gas explosion includes alleviating spleen atrophy, reducing congestion and bleeding in the red pulp of the spleen, and / or reducing spleen inflammation.

[0010] The beneficial effects of the above technical solution are as follows: by comparing with the control group, it was found that the spleen of the rat model of gas explosion injury was damaged, with multiple inflammatory exudates, hemorrhages and decreased spleen index. The spleen damage caused by gas explosion can be alleviated by relieving the above symptoms, which lays the foundation for studying the pathogenesis of spleen injury caused by gas explosion and provides a treatment basis for the early and reasonable treatment of spleen injury caused by gas explosion in clinical practice.

[0011] As a further improvement, the reduction of spleen inflammation includes downregulating the expression of IL-6 and ROS, and increasing the expression of IL-10, HMOX1, Nrf2, HO-1, COX2 and GPX4.

[0012] The beneficial effects of the above technical solution are as follows: This invention experimentally demonstrates that the spleen of a rat model of gas explosion injury exhibits severe inflammatory response, with upregulated expression of inflammatory factors such as IL-6 and IL-10, damage markers ROS, oxidative stress regulators HMOX1, and pathway-related genes Nrf2, HO-1, COX2, and GPX4. Furthermore, tert-butylhydroquinone significantly downregulated the expression of inflammatory factor IL-6 and damage marker ROS in the spleen of the gas explosion-induced rat model, and significantly upregulated the expression of anti-inflammatory factor IL-10, oxidative stress regulators HMOX1, and pathway-related genes Nrf2, HO-1, COX2, and GPX4. This indicates that tert-butylhydroquinone may regulate the oxidative stress injury process of the rat spleen induced by gas explosion through the Nrf2-HO1 signaling pathway, reducing the inflammatory response and thus alleviating the degree of spleen damage caused by gas explosion. This provides a direction for exploring new therapeutic targets for gas explosion-induced spleen injury and helps to elucidate the molecular mechanism by which oxidative stress alleviates gas explosion-induced spleen damage in rats.

[0013] As a further improvement, the formulation is a liquid dosage form.

[0014] The beneficial effects of the above technical solution are: liquid dosage form is more suitable for intraperitoneal injection, and intraperitoneal injection can deliver the preparation directly to the site of spleen damage in the shortest time, reaching the affected area directly while shortening the time for the drug to take effect. At the same time, this method of administration is more beneficial to patients who cannot take oral medication due to gas explosion.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] This invention successfully established an animal model of gas explosion injury (model group) using a real tunnel environment, laying the foundation for research on spleen injury caused by gas explosion. The study found that, compared with the normal control group, the model group rats exhibited severe splenic atrophy and inflammatory hemorrhage within the spleen. The expression of inflammatory factors such as IL-6 and IL-10, as well as damage markers ROS and oxidative stress regulators HMOX1 and pathway-related genes Nrf2, HO-1, COX2, and GPX4, was significantly upregulated, indicating that oxidative stress plays a crucial role in spleen injury caused by gas explosion.

[0017] This invention, through intraperitoneal injection of tert-butylhydroquinone (TBHQ) into a rat model of gas explosion injury immediately after a gas explosion, revealed that compared to the model group, rats injected with TBHQ showed a significantly increased spleen index, markedly improved splenic hemorrhage and inflammation, significantly decreased expression levels of the inflammatory factor IL-6 and the damage marker ROS, and significantly increased expression levels of the anti-inflammatory factor IL-10, the oxidative stress regulator HMOX1, and pathway-related genes Nrf2, HO-1, COX2, and GPX4. This indicates that TBHQ may regulate the oxidative stress injury process of the rat spleen induced by gas explosion through the Nrf2-HO1 signaling pathway, reducing the inflammatory response and thus alleviating the severity of gas explosion-induced spleen injury. This invention lays the foundation for elucidating the molecular mechanism by which oxidative stress alleviates gas explosion-induced spleen injury in rats and provides a new therapeutic target for gas explosion-induced spleen injury. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the actual tunnel structure of the present invention;

[0019] Figure 2 This is a schematic diagram of the front structure of the niche box of the present invention;

[0020] Figure 3 This is a schematic diagram of the cage structure of the present invention;

[0021] Figure 4 The spleen index of rats in different treatment groups in Example 2 of this invention;

[0022] Figure 5 The spleen morphology (200×) of rats in different treatment groups in Example 2 of this invention;

[0023] Figure 6 The expression of inflammatory factors in rats under different treatment groups in Example 3 of the present invention (where * represents P<0.05, ** represents P<0.01, and ns represents no difference);

[0024] Figure 7The expression of damage factors in rats under different treatment groups in Example 3 of the present invention (where * represents P<0.05, ** represents P<0.01, and ns represents no difference);

[0025] Figure 8 The expression changes of genes and proteins related to the oxidative stress pathway in the spleen of rats in different treatment groups in Example 4 of this invention (where * represents P<0.05, ** represents P<0.01);

[0026] In the picture:

[0027] 1-Horizontal tunnel; 11, 12-Sealing rings; 2-Inclined tunnel; 3-Cage; 31-Box body; 311-Reinforcing steel strips; 312-First layer; 313-Second layer; 314-Third layer; 315-Left bottom side edge of box body; 316-Right bottom side edge of box body; 32-Support body; 321-Left side edge of support body; 322-Right side edge of support body; 323-Left diagonal bar; 324-Right diagonal bar; 325-Front bar; 4-Detonation chamber; 5-Nest box; 51-Steel door; 52-Threaded hole; 53-Handle; 54-Fixing clip; 55-Bolt; 6-Pressure sensor; 7-Flame sensor. Detailed Implementation

[0028] In this invention, the roadway is designed based on a simulation of an underground gas explosion environment in a coal mine. The fixed cages are positioned at 160m and 240m. The roadway diameter is 3m and the length is 800m. A schematic diagram of the actual roadway structure is shown below. Figure 1 A schematic diagram of the front structure of the niche box is shown below. Figure 2 The cage structure diagram is as follows: Figure 3 Based on the preliminary experimental results of our team, the explosion parameters are as follows: 100m³ of mixed gas in a confined space. 3 The oxygen concentration is 8%–20%, the methane concentration is 9%, and the central combustion pressure is 1.6–2.0 kg. The process includes the following steps:

[0029] Step 1: Set up the actual tunnels.

[0030] like Figure 1 This is a schematic diagram of the actual tunnel structure involved in the gas explosion of this invention. Figure 1As shown, the actual tunnel used in this experiment is an arched tunnel, consisting of a horizontal level tunnel 1 and an upwardly sloping inclined tunnel 2. The end of the level tunnel 1 is a blind end, and the end of the inclined tunnel 2 opens to the ground surface. A detonation chamber is set at the blind end of the level tunnel. During setup, a sealing ring 11 is circumferentially installed on the side wall of the blind end of the level tunnel. A plastic film is then sealed and clamped onto the sealing ring 11, forming a detonation chamber 4 between the blind end of the level tunnel and the plastic film. (Note: Sealing rings 11 and 12, on the one hand, prevent the gas from causing toxic damage to the experimental animals before detonation, thus affecting the results of the explosion experiment; on the other hand, using a plastic film to separate the detonation chamber, the low strength of the plastic film will not excessively weaken the blast shock wave, thus avoiding excessive experimental errors. Depending on the different explosion yield requirements of different experiments, sealing rings at different positions with plastic film can be used to form detonation chambers of different sizes at the blind end of the level tunnel, meeting the gas volume requirements for different explosion yields.)

[0031] Step 2: Set up the niche box and sensor.

[0032] A niche box 5 is installed on the side wall of the level lane 1, such that the height of the niche box 5 from the ground of the level lane 1 is 40-50cm, and the placement depth of the niche box in the level lane is 40cm. Figure 2 This is a schematic diagram of the front structure of the niche box 5 in a gas explosion according to the present invention. The niche box 5 is equipped with a steel door 51, and a threaded hole 52 is provided in the middle of the steel door 51. A flame sensor 7 and a pressure sensor 6 are installed in the niche box 5, with the probes of the flame sensor 7 and pressure sensor 6 extending out of the steel door 51 through the threaded hole 52. A handle 53 can be provided on the steel door 51 for easier opening and closing. Fixing clips 54 can also be installed on the sidewall of the tunnel around the steel door 51, and bolts 55 can be used to fix the steel door 51 to the fixing clips 54. This can better protect the flame sensor and pressure sensor in the niche box 5, ensuring they remain intact during an explosion. Multiple niche boxes are installed along the direction of the horizontal tunnel, spaced 10m-20m apart, to better collect pressure and flame data during an explosion. Flame and pressure sensors are connected to an external monitoring system to test and record the magnitude of the shock wave pressure and the spread of the flame in the tunnel during the explosion, thus helping to study the relationship between animal injuries and the magnitude of the shock wave pressure and the flame.

[0033] Step 3: Set up cages and place the experimental animals in the cages.

[0034] Figure 3This is a schematic diagram of the cage structure in a gas explosion according to the present invention. When setting up the cage, the cage 3 is fixedly installed on the ground of the level tunnel 1. The cage 3 includes a box body 31 and a support body 32. The box body 31 is a cuboid frame structure, with all edges of the box body 31 made of steel. Each face of the box body 31 is enclosed by a steel mesh with a mesh diameter of 3-5 cm. The support body 32 is located in front of the box body 31 and includes a left side edge 321, a right side edge 322, a diagonal rod, and a front rod 325. The left bottom side edge 315 of the box body extends towards the front of the box body to form the left side edge 321 of the support body, and the right bottom side edge 316 of the box body extends towards the front of the box body to form the right side edge 322 of the support body. The left side edge 321 and the right side edge 322 of the support body are of equal length. The two ends of the front rod 325 are respectively connected to the ends of the left side edge 321 and the right side edge 322 of the support body. The cage is fixed in place; the diagonal brace includes a left diagonal brace 323 and a right diagonal brace 324. The left diagonal brace 323 is fixedly connected to the end of the left side edge 321 of the support body and the top of the left side edge of the front surface of the box body; the right diagonal brace 324 is fixedly connected to the end of the right side edge 322 of the support body and the top of the right side edge of the front surface of the box body 31; the lengths of the left side edge 321 and the right side edge 322 of the support body are greater than the lengths of the left bottom side edge 315 and the right bottom side edge 316 of the box body; a reinforcing steel strip 311 is provided on the front surface of the box body 31. When installing the cage, the support body 32 of the cage 3 is oriented towards the detonation chamber 4. The cage in this invention has three layers. SD rats are placed on the first layer 312, the second layer 313 and the third layer 314 respectively. The SD rats are placed under anesthesia in accordance with ethical requirements.

[0035] Cage 3 can be placed every 40 meters along the direction of level tunnel 1, i.e., one cage at each of six distances: 40m, 80m, 120m, 160m, 200m, and 240m, to simulate the blast injuries of miners at different distances from the explosion source in a coal mine. Preliminary experiments with cages placed at different locations showed that placing cages at 160m resulted in an acceptable mortality rate for rats, and the spleen damage caused by the explosion model was easy to observe and study, making it suitable for establishing a gas explosion model.

[0036] Step 4: Detonate.

[0037] The detonation chamber 4 was filled with 100m³ of gas at a concentration of 9.0%. 3 The gas is a uniformly mixed gas with an oxygen concentration of 8% to 20%. It is ignited by an ignition system using two ignition heads as ignition sources. Each ignition head has an energy of 10J and a central combustion pressure of 1.6 to 2.0kg, causing the gas to explode and propagate in the detonation chamber.

[0038] After the actual gas explosion in the tunnel was completed and ventilation was completed, the experimental animals were quickly transferred out of the tunnel in animal cages, and the rats in each treatment group were promptly treated accordingly.

[0039] The specific experimental method for gas explosion can be found in Chinese invention patent application publication number CN112051303A.

[0040] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto; however, these embodiments are merely examples and do not constitute any limitation on the scope of the present invention. Modifications or substitutions to the details and form of the present invention can be made without departing from the spirit and scope of the present invention, but such modifications and substitutions all fall within the scope of protection of the present invention. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods. Unless otherwise specified, the experimental materials used in the following embodiments are all purchased from conventional biochemical reagent manufacturers.

[0041] The following is a brief introduction to some of the biological materials, experimental reagents, and experimental equipment involved in the following examples and experimental cases:

[0042] Instruments and equipment:

[0043] CIT-2MK Infrared Linearized Temperature Sensor (Institute of Automation, Chinese Academy of Sciences): Temperature range: 400~2000℃, Response time: 67ms, Applicable range: 0~60℃; CYG Series Solid-State Piezoresistive Pressure Sensor (Baoji Sensor Research Institute, Shaanxi Qinming Electronics Group Co., Ltd.): Ranges are 0.2MPa and 0.1MPa respectively, Applicable range: -40~80℃, Output signal range: 0~5V; Flame Sensor (Baoji Sensor Research Institute, Shaanxi Qinming Electronics Group Co., Ltd.); CS20000 Dynamic Testing and Analysis System (Chengdu Taist Electronic Information Co., Ltd.); BX60 Fluorescence Microscope (Olympus Corporation, Japan); 120A Electronic Analytical Balance (Drecisa, Switzerland); LS-O410 Constant Temperature Oven (Fisher Scientific, USA); OFT-100 Large and Small Mouse Opening Activity Experiment System (Chengdu Taimeng Software Co., Ltd.); Gas Explosion Large-Scale Roadway Test System (Chongqing Research Institute of China Coal Technology & Engineering Group Co., Ltd.).

[0044] Reagents:

[0045] All ELISA kits are from the brand abclonal, including IL-6 (catalog number: RK00020), IL-10 (catalog number: RK00050), ROS (catalog number: RK15283), and HMOX1 (catalog number: RK03723).

[0046] Laboratory animals and culture conditions:

[0047] Male SD rats, weighing 180-220g, were provided by the Experimental Animal Center of Daping Hospital, Army Medical University. The rats were housed in an animal room with a room temperature maintained at 24±1℃, relative humidity at 65%, and a 12 / 12h light / dark cycle. The daily housing environment met the relevant requirements of GB 14925—2010 "Experimental Animal Environment and Facilities".

[0048] Statistical analysis:

[0049] The experimental data of this invention were analyzed and plotted using GraphPad Prism X7 software. Experimental results are expressed as mean ± standard error (mean ± SEM). Comparisons between two groups were performed using t-tests, and comparisons among multiple groups were performed using one-way ANOVA. A p-value < 0.05 was considered statistically significant.

[0050] I. Specific Examples of the Application of Tert-Butylhydroquinone in the Preparation of Agents for Mitigating Spleen Damage Caused by Gas Explosions

[0051] Example 1: Establishment and treatment of a rat gas explosion model

[0052] One hundred and twenty healthy male SD rats were randomly divided into four groups according to body weight: control group (NC group), gas explosion model group (Model group), Nrf2 inhibitor group (Model+ATRA group), and Nrf2 activator group (Model+TBHQ group), with ten rats in each group. They were further divided into groups at 24h, 3d, and 7d post-explosion, with ten rats in each group. In the Model+ATRA group, rats were administered ATRA 5 mg / kg intraperitoneally once daily after the gas explosion model was established. In the Model+TBHQ group, rats were administered TBHQ 12.5 mg / kg intraperitoneally once daily after the gas explosion model was established. The NC and Model groups received an equal volume of DMSO solvent. All rats in each group were anesthetized, weighed, and had blood drawn from the abdominal aorta at 24h, 3d, and 7d, before being sacrificed and their spleen tissue was collected for further research.

[0053] Example 2: Evaluation of the degree of spleen damage in a gas explosion

[0054] 1. Detection of spleen tissue index in rats

[0055] This embodiment uses changes in the spleen index of rats to directly reflect the degree of spleen atrophy and indirectly reflect the state of spleen immune function. The experimental rats were weighed at specified time points, then sacrificed by cervical dislocation, and the intact spleens were removed and weighed. Spleen index = spleen mass (mg) / body weight (g).

[0056] Effects of different treatment groups on spleen index in rats, such as Figure 4As shown in the figure, the spleen index of rats at 24h, 72h, and 7 days after the explosion was 46.3%, 52.9%, and 61.9% of the normal group, respectively. After treatment with the inhibitor ATRA, the spleen index at 24h, 72h, and 7 days after the explosion was 40.3%, 37.5%, and 40.6% of the normal group, respectively. After treatment with the agonist TBHQ, the spleen index at 24h, 72h, and 7 days after the explosion was 49.3%, 60.3%, and 68.9% of the normal group, respectively. The results of the spleen index detection indicate that the spleen of rats suffered severe atrophy after the gas explosion, and the Nrf2 activator TBHQ could inhibit spleen atrophy, indicating that it has the effect of reducing immune function damage.

[0057] 2. HE staining

[0058] Rats in each treatment group were euthanized under abdominal aortic anesthesia, and spleen tissue was harvested, fixed in 4% paraformaldehyde, and stored in liquid nitrogen for pathological section preparation. The spleen tissue was embedded in paraffin, and the paraffin blocks were cut into 3-4 μm thin slices. The slices were copied at 60°C for 60 min, dewaxed using a xylene gradient and hydrated using an alcohol gradient, stained with hematoxylin and eosin (HE), and mounted. The degree of spleen tissue damage was observed under a 200x optical microscope.

[0059] HE staining results showed that the spleen structure of rats in the control group was normal and intact, with neatly arranged cells and full, intact nuclei. The red pulp (RP) and white pulp (WP) were clearly demarcated, and the white pulp contained abundant, densely arranged lymphocytes. In the model group, inflammatory hemorrhage was observed in the spleen tissue, with reduced and loosely arranged lymphocytes, blurred cell structure, and some cells showing nucleus loss and diffuse necrosis. Compared with the model group, the inhibitor group showed more severe spleen damage with multiple sites of inflammatory cell infiltration. The activator group showed reduced spleen damage, with decreased congestion and hemorrhage in the red pulp. Figure 5 .

[0060] Example 3: Changes in inflammatory factors and damage markers in the spleen of rats in each treatment group

[0061] This embodiment uses enzyme-linked immunosorbent assay (ELISA) to detect the expression of inflammatory factors IL-6, anti-inflammatory factor IL-10, damage marker ROS, and oxidative stress regulator HMOX1 in the spleen of rats in each treatment group. Spleen tissue from each group was collected, homogenized with lysis buffer, centrifuged at 12,000 rpm for 10 min at 4°C, and the supernatant was collected. The ELISA procedure was strictly performed according to the kit instructions to detect the expression of each factor. A standard curve was prepared by measuring the OD values ​​of different concentrations of standards using a microplate reader (wavelength 450 nm) to calculate the levels of relevant factors in the samples.

[0062] The expression levels of inflammatory factors, such as Figure 6 As shown, the levels of IL-6 and IL-10 in the spleen of rats in the model group were significantly higher than those in the control group (P < 0.01). Compared with the model group, the expression level of IL-6 in the inhibitor group was significantly increased (P < 0.05), while the expression level of IL-10 did not change significantly at 24 h, but decreased significantly at 72 h and 7 d. The agonist TBHQ could significantly downregulate the expression of the inflammatory factor IL-6 (P < 0.05) and upregulate the expression of the anti-inflammatory factor IL-10 (P < 0.01).

[0063] Expression levels of damage markers, such as Figure 7 As shown, the levels of the injury marker ROS and the oxidative stress regulator HMOX1 in the spleen of rats in the model group were significantly higher than those in the control group (P < 0.05); the expression level of ROS in the inhibitor group was significantly higher than that in the model group (P < 0.05), and the expression level of HMOX1 was significantly lower (P < 0.05); while the expression of ROS in the agonist group was significantly lower than that in the model group, and the expression level of HMOX1 was significantly higher (P < 0.05).

[0064] Note: HMOX1 belongs to the heme oxygenase family and is closely related to many physiological and pathological processes. When cells are subjected to oxidative stress, HMOX1 protein expression gradually increases, inhibiting the expression of inflammatory factors such as tumor necrosis factor and interleukin-1, thereby suppressing inflammation and exerting a protective effect.

[0065] Example 4: The Nrf2 / HO-1 signaling pathway protects against oxidative stress damage during gas explosions.

[0066] 1. Quantitative Real-Time PCR (qRT-PCR) Detection

[0067] 200 mg of spleen tissue from rats in each treatment group was weighed, rapidly ground in liquid nitrogen, and total RNA was extracted using Trizol (Invitrogen, Carlsbad, USA). RNA concentration was determined according to PrimeScript. TM cDNA was obtained by reverse transcription using the RT Master Mix kit. Real-time quantitative PCR was performed according to the Step One Plus Real Time PCR System kit steps to detect the fluorescence signal values ​​of each gene amplification product, and the relative expression levels of genes were calculated using β-actin as an internal reference. The qRT-PCR conditions were: 95℃ for 30 min, 95℃ for 5 s, 60℃ for 30 s, for 40 cycles. The obtained data were analyzed using 2... －ΔΔCt Relative quantitative analysis was performed. Each sample was tested three times. Primers used in the experiment were designed using Primer Premier 5.0 software and synthesized by Sangon Biotech Co., Ltd. (Shanghai, China), as detailed in Table 1.

[0068] Table 1 Primer sequences

[0069]

[0070] 2. Western blot detection

[0071] 200 mg of spleen tissue was weighed and added to 1 mL of RIPA protein lysis buffer. The tissue suspension was homogenized using an ultrasonic mixer, lysed on ice for 30 min, centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant was collected. Protein quantification was performed using a BCA kit. After SDS-PAGE electrophoresis, the target protein was transferred to a PVDF membrane, blocked with 5% skim milk powder, washed with TBST, and incubated overnight at 4 °C with primary antibodies Nrf2, HO-1, COX2, and GPX4 (dilution ratio: 1:1000). The membrane was then incubated with secondary antibody at room temperature for 1 hour, followed by BCIP / NBT color development (Sangon Biotech, Shanghai) using an automatic exposure machine. β-actin was used as an internal control. The gray values ​​of each target band and the internal control were calculated using computer software. All experiments were performed in triplicate.

[0072] Nrf2 plays an important role in protecting cells from oxidative stress. Western blot and qRT-PCR results are as follows: Figure 8 As shown. By Figure 8 It was found that the expression of Nrf2 and HO-1 proteins and mRNAs was increased in the model group; however, compared with the activator group, the levels of Nrf2 and HO-1 proteins and mRNAs were significantly inhibited in the inhibitor group.

[0073] In summary, this invention successfully established an animal model of gas explosion injury using a real tunnel environment, exploring from a new perspective whether the Nrf2 / ARE signaling pathway plays an antioxidant protective role in gas explosion-induced spleen tissue damage. Experimental results demonstrated that the model group rats exhibited severe inflammatory responses in the spleen, with upregulated expression of inflammatory factors such as IL-6 and IL-10, damage markers ROS, oxidative stress regulator HMOX1, and pathway-related genes Nrf2, HO-1, COX2, and GPX4. Conversely, the TBHQ-induced group rats showed significantly downregulated expression levels of the inflammatory factor IL-6 and the damage marker ROS, and significantly upregulated expression levels of the anti-inflammatory factor IL-10, the oxidative stress regulator HMOX1, and pathway-related genes Nrf2, HO-1, COX2, and GPX4. TBHQ may regulate the oxidative stress injury process of the rat spleen induced by gas explosion through the Nrf2-HO1 signaling pathway, reducing the inflammatory response and thus alleviating the severity of spleen damage caused by gas explosion in rats. Future research will focus on elucidating the molecular mechanism by which oxidative stress alleviates spleen damage in rats caused by gas explosions, aiming to provide new targets for the treatment of spleen damage caused by gas explosions.

[0074] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of tert-butylhydroquinone in the preparation of agents for mitigating spleen damage caused by gas explosions.

2. The application of tert-butylhydroquinone according to claim 1 in the preparation of an agent for mitigating spleen damage caused by gas explosion, characterized in that: The measures to mitigate spleen damage caused by gas explosions include relieving spleen atrophy, reducing congestion and bleeding in the red pulp of the spleen, and / or reducing spleen inflammation.

3. The application of tert-butylhydroquinone according to claim 2 in the preparation of an agent for mitigating spleen damage caused by gas explosion, characterized in that: The reduction of spleen inflammation includes downregulating the expression of IL-6 and ROS, and increasing the expression of IL-10, HMOX1, Nrf2, HO-1, COX2 and GPX4.

4. The use of tert-butylhydroquinone according to any one of claims 1 to 3 in the preparation of an agent for mitigating spleen damage caused by gas explosion, characterized in that: The formulation is a liquid dosage form.

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