Application of dimethyl fumarate and signaling pathway thereof in inhibition of arsenic-induced rat liver and kidney immune inflammatory response

Abstract

The invention belongs to the technical field of biological medicine, and provides dimethyl fumarate and application of a signal channel thereof in inhibition of arsenic-induced rat liver and kidney immune inflammation reaction. According to the present invention, with the application of the sodium arsenite in the rat liver and kidney immune inflammation reaction, the activation of the HMGB1 / TLR4 / NF-kappa B signal channel is inhibited through the intervention of the dimethyl fumarate, the Th1 / Th2 / Th17 / Treg balance disorder caused by the sodium arsenite and the CD4 + T cell subset differentiation disorder are antagonized, and the arsenic-induced rat liver and kidney immune inflammation reaction is alleviated; a rat subchronic arsenic exposure model is established, dimethyl fumarate is adopted for intervention, arsenic-induced liver and kidney tissue immune inflammatory response and CD4 + T cell subset differentiation disorder are discussed on the basis of an HMGB1 / TLR4 / NF-kappa B signal channel, and the antagonism and potential action mechanism of dimethyl fumarate in arsenic-induced rat immune injury are preliminarily clarified. And a theoretical basis and a research thought are provided for arsenic poisoning prevention and treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of dimethyl fumarate and its signal pathway in inhibiting arsenic-induced hepatic and renal immune inflammatory response in rats; more specifically, dimethyl fumarate DMF inhibits the activation of HMGB1 / TLR4 / NF-κB signal pathway, antagonizes the Th1 / Th2 / Th17 / Treg balance disorder caused by sodium arsenite, and improves CD4 + T cell subset differentiation disorder reduces arsenic-induced immune inflammatory response in the liver and kidneys of rats. Background Art

[0002] Inorganic arsenic is a metalloid element that is widely present in nature. It is classified as a Class I carcinogen by the International Agency for Research on Cancer and poses a major threat to global health. It is estimated that between 94 million and 220 million people may be exposed to high-arsenic groundwater, the vast majority of whom (94%) are in Asia[1]. The immune system is one of the targets of arsenic poisoning. Arsenic can affect innate immunity and specific immune responses[2], and impair the normal physiological functions of a variety of immune cells, such as regulating CD4 + Differentiation of Th1, Th2, Th17 and Treg in T cell subsets[3]. The liver and kidney are the main target organs of arsenic poisoning, and they play an important role in arsenic metabolism and excretion, respectively[4]. However, the toxic mechanism of arsenic-induced liver and kidney immune damage is still unclear.

[0003] High mobility group box 1 protein (HMGB1) is an important member of damage associated molecular patterns (DAMPs). It is actively secreted or passively released outside the cell when the body's own cells are damaged or necrotic, and plays an important role in the occurrence and development of sepsis, shock, autoimmune diseases and chronic inflammation[5].

[0004] Toll-like receptor 4 (TLR4), as an important type of pattern recognition receptor (PRR), is a “bridge” connecting the body’s innate immunity and specific immune response[6]. It can recognize HMGB1 and activate the NF-κB signaling pathway through a MyD88-dependent pathway to promote inflammatory response[7]. Currently, the interaction between TLR4 and DAMP is used as a starting point to explore the mechanism of arsenic activating the body’s innate immune system and inducing the body’s immune regulation effect, but related research is still rare.

[0005] Dimethyl fumarate (DMF) is a new drug that can be taken orally to treat multiple sclerosis. Recent studies have shown that DMF has therapeutic potential for a variety of diseases besides multiple sclerosis. Studies have shown that it has the effects of protecting nerves, regulating immunity, and reducing oxidative stress [8]. However, the mechanism of how DMF regulates the function of the immune system has not been fully elucidated. Some studies have found that it may be related to DMF's ability to inhibit Toll-like receptor activation and the downstream NF-κB signaling pathway, thereby exerting an anti-inflammatory effect [9]. However, its role in arsenic-induced immune damage has not been reported. Summary of the invention

[0006] The present invention provides an application of dimethyl fumarate and its signal pathway in inhibiting arsenic-induced hepatic and renal immune inflammatory response in rats. The present invention establishes a subchronic arsenic exposure model in rats and uses dimethyl fumarate for intervention, and explores the arsenic-induced hepatic and renal immune inflammatory response and CD4+ TLR4 / NF-κB signal pathway based on the HMGB1 / TLR4 / NF-κB signal pathway. + The differentiation disorder of T cell subsets was also eliminated, and the antagonistic effect of dimethyl fumarate on arsenic-induced liver and kidney immune damage in rats and its potential mechanism of action were preliminarily elucidated, in order to provide a theoretical basis and research ideas for the prevention and treatment of arsenic poisoning.

[0007] The present invention is realized by the following technical scheme: application of dimethyl fumarate in inhibiting arsenic-induced hepatic and renal immune inflammatory response in rats, wherein the DMF intervention inhibits activation of HMGB1 / TLR4 / NF-κB signaling pathway, antagonizes Th1 / Th2 / Th17 / Treg balance disorder and CD4 + T cell subset differentiation disorder reduces arsenic-induced immune inflammatory response in the liver and kidneys of rats.

[0008] Furthermore, the DMF intervention reduces the secretion of inflammatory factors IL-1β, IL-6 and TNF-α, and inhibits the inflammatory response.

[0009] The DMF intervention caused changes in the expression levels of Th1 characteristic cytokine IFN-γ and transcription factor T-bet, Th2 characteristic cytokine IL-4 and transcription factor GATA-3, Th17 characteristic cytokine IL-17 and transcription factor ROR-γt, and Treg characteristic cytokine IL-10 and transcription factor Foxp3.

[0010] The present invention also provides the DMF intervention, which down-regulates the TLR4 protein level in liver and kidney tissues and the MyD88, NF-κB p65 and TRAF6 protein levels in the kidneys.

[0011] Furthermore, the DMF intervention is intragastric administration of 60 mg / kg DMF once a day.

[0012] The present invention verifies that subchronic arsenic exposure causes immune inflammatory response and immunotoxicity in the liver and kidney, which may be related to the increased release of HMGB1 induced by arsenic exposure, activation of TLR4 / NF-κB inflammatory pathway, and disturbance of Th1 / Th2 / Th17 / Treg balance, CD4 + The results of this study are related to the differentiation disorder of T cell subsets. DMF may reduce the liver and kidney damage caused by arsenic exposure by reducing the level of proinflammatory factors and regulating the balance of Th1 / Th2 / Th17 / Treg. The potential mechanism may be related to DMF's ability to inhibit the activation of the HMGB1 / TLR4 / NF-κB pathway. The results obtained in this paper will provide useful research clues and theoretical basis for the clinical application of dimethyl fumarate to inhibit the immunotoxicity of arsenic. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Results are the weight changes of rats; Figure 2 HE staining of rat liver tissue; in the figure: A is the control group (×100); B is 10 mg / L NaAsO 2 Group (×100); C is 25 mg / L NaAsO 2 Group (×100); D is 50 mg / L NaAsO 2 group (×100); E is the DMF intervention group (×100); F is the control group (×400); G is 10 mg / L NaAsO 2 Group (×400); H is 25 mg / L NaAsO 2 Group (×400); I is 50 mg / L NaAsO 2 group (×400); J is the DMF intervention group (×400); Figure 3 HE staining of rat kidney tissue; in the figure: A is the control group (×100); B is 10 mg / L NaAsO 2 Group (×100); C is 25 mg / L NaAsO 2 Group (×100); D is 50 mg / L NaAsO 2 group (×100); E is the DMF intervention group (×100); F is the control group (×400); G is 10 mg / L NaAsO 2 Group (×400); H is 25 mg / L NaAsO 2 Group (×400); I is 50 mg / L NaAsO 2 group (×400); J is the DMF intervention group (×400); Figure 4 Middle: A is rat liver CD4 +Expression of characteristic cytokines of T cell subsets; B is rat liver CD4 + Expression of characteristic transcription factors of T cell subsets; Note: Compared with the control group, a P <0.05; with 50mg / L NaAsO 2 Compared with the poisoned group, b P <0.05; Figure 5 Middle: A is rat kidney CD4 + Expression of characteristic cytokines of T cell subsets; B is CD4 + Expression of characteristic transcription factors of T cell subsets; Note: Compared with the control group, a P <0.05; with 50mg / L NaAsO 2 Compared with the poisoned group, b P <0.05; Figure 6 The expression of proteins related to HMGB1 / TLR4 / NF-κB pathway in rat liver; compared with the control group, *a P <0.05; in the figure: A is the rat liver control group, 10mg / L NaAsO 2 Group, 25mg / L NaAsO 2 Group, 50mg / L NaAsO 2 Histone expression electrophoresis; B is the rat liver control group, 10mg / L NaAsO 2 Group, 25mg / L NaAsO 2 Group, 50mg / L NaAsO 2 Histone quantitative analysis; C is the rat liver control group, 50mg / L NaAsO 2 Electrophoresis of protein expression in rat liver of control group and DMF intervention group; D is the electrophoresis of protein expression in rat liver of control group and 50mg / L NaAsO 2 Group and DMF intervention histone quantitative analysis; Figure 7 The expression of proteins related to HMGB1 / TLR4 / NF-κB pathway in rat kidney; Note: Compared with the control group, *a P <0.05; with 50mg / L NaAsO 2 Compared with the poisoned group, b P <0.05; in the figure: A is the rat kidney control group, 10mg / L NaAsO 2 Group, 25mg / L NaAsO 2 Group, 50mg / L NaAsO2 Histone expression electrophoresis; B is the rat kidney control group, 10mg / L NaAsO 2 Group, 25mg / L NaAsO 2 Group, 50mg / L NaAsO 2 Histone quantitative analysis; C is the rat kidney control group, 50mg / L NaAsO 2 Electrophoresis of protein expression in rat kidneys of control group and DMF intervention group; D is the electrophoresis of protein expression in rat kidneys of control group and 50 mg / L NaAsO 2 Quantitative analysis of histones in the DMF-treated and DMF-treated groups. DETAILED DESCRIPTION

[0014] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and the disclosure and materials cited therein are hereby incorporated by reference.

[0016] Technical equivalents to the specific embodiments described that are apparent to those skilled in the art using no more than routine experimentation are intended to be encompassed by this application.

[0017] The experimental methods in the following examples are conventional methods unless otherwise specified. The instruments and equipment used in the following examples are conventional laboratory instruments and equipment unless otherwise specified; the experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified.

[0018] 1. Materials and Methods 1. Main reagents and instruments: Sodium arsenite (Sigma, USA), dimethyl fumarate (Shanghai Yien Chemical Technology Co., Ltd.), BCA protein quantification kit (Wuhan Boster Biotechnology Co., Ltd.), IL-1β, IL-6, TNF-α enzyme-linked immunosorbent assay kit (Jiangsu Enzyme Immunity Industry Co., Ltd.), RNAiso Plus, PrimeScript TM FAST RT Reverse Transcription Kit, TB Green Premix Ex Taq TMII Real Time PCR kit (TaKaRa Bio Co., Ltd., Japan), HMGB1, TLR4, MyD88, MD2, NF-κB p65, TRAF6, β-actin antibodies (Wuhan Abotek Biotechnology Co., Ltd.), electrophoresis instrument (DYY-6C, Beijing Liuyi Biotechnology Co., Ltd.), fluorescence quantitative PCR instrument (CFX Connect, Bio-Rad, USA), enzyme label detector (Tecan Infinite M200 Pro, Tecan, Switzerland), chemiluminescence imaging system (ChemiDoc™ MP, Bio-Rad, USA).

[0019] 2. Experimental animals and treatment: 40 SD female rats were selected and, after one week of adaptive feeding, were randomly divided into 5 groups according to body weight, with 8 rats in each group. 2 ), low-dose group (10 mg / L NaAsO 2 ), medium dose group (25 mg / L NaAsO 2 ), high-dose group (50 mg / L NaAsO 2 ) and dimethyl fumarate intervention group (50 mg / LNaAsO 2 +60mg / kg DMF). The animals were exposed to water freely for 12 weeks, and the dimethyl fumarate intervention group was given 60mg / kg / d DMF by gavage once a day. This study was approved by the Ethics Committee of Shanxi Medical University (No. 2021-314), and the experiment complied with animal ethics requirements.

[0020] 3. Hematoxylin-eosin (HE) staining: The liver and kidney tissues were fixed with 4% paraformaldehyde, dehydrated with gradient ethanol dehydration method, and embedded in paraffin. The sections were fixed on glass slides, stained with HE, and sealed with neutral gum. The pathological changes of rat liver and kidney tissues were observed under a microscope.

[0021] 4. Enzyme-linked immunosorbent assay (ELISA): Collect blood in a sterile tube, centrifuge at 2500r / min for 20min at 4℃, and collect the supernatant. Follow the instructions of the ELISA kit to add samples, incubate, wash, add enzyme-labeled reagent, incubate and wash again, add color developer for 10min, and terminate the reaction. Measure the absorbance at 450nm to determine the actual concentrations of IL-1β, IL-6, and TNF-α.

[0022] 5. qRT-PCR detection of mRNA expression level: The total RNA of liver and kidney tissues was extracted by Trizol method, and the RNA was reverse transcribed into cDNA using a reverse transcription kit and stored in a -80℃ refrigerator for later use. The reaction system (10μl) was prepared according to the kit instructions: TB Green Premix Ex Taq Ⅱ 5.0μl, forward and reverse primers 0.4μl each, cDNA 0.8μl, ddH 2 O3.4μl. The PCR reaction was carried out in two steps, with the following conditions: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing for 30s, for a total of 40 cycles. GAPDH was used as the internal reference gene, and 2 -ΔΔCt The relative expression level of the target gene was calculated.

[0023] 6. Western blot detection of protein expression level: extract total protein from liver and kidney tissues, use BCA method to detect protein concentration, and heat at 100℃ or 37℃ for 10 min to fully denature the protein.

[0024] According to the molecular weight of the target protein, 8%, 10% or 12% separation gel and 5% concentration stacking gel were prepared, 30 μg protein was loaded into each well, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed. The gel protein bands were wet-transferred to NC membranes. The membranes were placed in TBST solution containing 5% skim milk powder and blocked at room temperature for 90 min. The membranes were incubated with primary antibodies of HMGB1 (1:1000), TLR4 (1:2000), MyD88 (1:1000), MD2 (1:1000), NF-κB p65 (1:1000), TRAF6 (1:1000), and β-actin (1:10000) at 4°C overnight. After washing the membranes, HRP-labeled goat anti-rabbit IgG (1:10000) and goat anti-mouse IgG (1:10000) were added and incubated at room temperature for 60 min. The membranes were washed with TBST solution for 3 times. Enhanced chemiluminescence reagent was used for color development. The gel imaging system was used for scanning and analysis. Image J software was used to quantitatively analyze the gray value of protein bands.

[0025] 7. Statistical analysis: SPSS 26.0 software was used for data processing. One-way analysis of variance was used for comparison among multiple groups. LSD method was used for pairwise comparison when variance was equal, and Tamhane T2 test was used for unequal variance. GraphPad Prism 9 software was used for plotting. P The difference was statistically significant when the value was <0.05.

[0026] 2. Experimental Results 1. Rat weight and liver and kidney coefficients: The results of rat weight changes are as follows: Figure 1The analysis of rat liver and kidney coefficients is shown in Table 1. The results showed that during the experiment, the rats ate and drank normally, had no hair loss, and were in good mental state. There was no significant change in the body weight of rats in each group, and the differences were not statistically significant ( P >0.05). There was no statistically significant difference in the liver and kidney coefficients ( P >0.05).

[0027] Table 1: Analysis of rat liver and kidney coefficients 2. Rat liver and kidney histopathology: HE staining results are as follows Figure 2 , Figure 3 The results showed that compared with the control group, 10 mg / L NaAsO 2 The liver lobule structure was damaged in the group, 25 mg / L NaAsO 2 The hepatocytes in the group showed fatty degeneration, and round vacuoles of different sizes were seen in the cytoplasm. 50 mg / L NaAsO 2 The liver tissue of the group was disordered, and inflammatory cell infiltration was observed in the liver lobules; NaAsO 2 The renal tubules in the poisoned group were atrophic, the structure was unclear, and the glomerular mesangial proliferation was observed. 25 and 50 mg / L NaAsO 2 The group was accompanied by a small amount of inflammatory cell infiltration. 2 Compared with the control group, the liver tissue boundaries in the DMF intervention group were slightly clearer and arranged relatively neatly; the kidney tissue damage was alleviated, and the inflammatory cell infiltration was improved.

[0028] 3. Expression levels of IL-1β, IL-6 and TNF-α in rat serum: The results are shown in Table 2. The results show that compared with the control group, 10 mg / L NaAsO 2 The TNF-α content in the group was increased, and the difference was statistically significant ( P <0.05, Table 2), 25, 50 mg / L NaAsO 2 The expression levels of IL-1β, IL-6, and TNF-α in the serum of the two groups were significantly increased, and the difference was statistically significant ( P <0.05); and 50mg / LNaAsO 2 Compared with the control group, the levels of IL-1β, IL-6, and TNF-α in the DMF intervention group were significantly reduced, and the difference was statistically significant ( P <0.05).

[0029] Table 2: Expression levels of inflammatory factors in rats Note: Compared with the control group, a P<0.05; with 50mg / L NaAsO 2 Compared with the poisoned group, b P <0.05.

[0030] 4. Rat CD4 + Expression levels of characteristic cytokines and transcription factors in T cell subsets Results in liver tissue Figure 4 As shown, compared with the control group, 50 mg / L NaAsO 2 The mRNA level of Th1 cytokine IFN-γ in the group was significantly increased, and the difference was statistically significant ( P <0.05); 25, 50 mg / L NaAsO 2 The mRNA level of Th1 transcription factor T-bet in the 44-hectare group was significantly increased, and the difference was statistically significant ( P <0.05); the mRNA levels of Th17 cytokine IL-17 and transcription factor ROR-γt increased with NaAsO 2 The difference was statistically significant ( P <0.05); each NaAsO 2 The mRNA levels of Th2 cytokine IL-4, transcription factor GATA-3, and Treg cytokine IL-10 in the exposure group decreased significantly, with statistically significant differences ( P <0.05).

[0031] With 50mg / L NaAsO 2 Compared with the control group, the mRNA levels of Th1 cytokine IFN-γ and transcription factor T-bet, Th17 cytokine T-bet and transcription factor ROR-γt in the DMF intervention group were significantly decreased, and the difference was statistically significant ( P <0.05), the mRNA level of GATA-3, a Th2 transcription factor, was significantly increased, and the difference was statistically significant ( P <0.05).

[0032] The results in kidney tissue were as follows Figure 5 As shown, compared with the control group, 50 mg / L NaAsO 2 The mRNA level of Th1 cytokine IFN-γ in the group was significantly increased, and the difference was statistically significant ( P <0.05); 25, 50 mg / L NaAsO 2 The mRNA level of Th1 transcription factor T-bet in the 44-hectare group was significantly increased, and the difference was statistically significant ( P <0.05); each NaAsO 2The mRNA levels of Th2 cytokine IL-4 and transcription factor GATA-3 in the exposure group decreased significantly, and the difference was statistically significant ( P <0.05); 50mg / L NaAsO 2 The mRNA level of Th17 cytokine IL-17 in the 4-group group was significantly increased, and the difference was statistically significant ( P <0.05); 25, 50 mg / L NaAsO 2 The mRNA level of Th17 transcription factor ROR-γt in the 44-hectare group was significantly increased, and the difference was statistically significant ( P <0.05); 50mg / L NaAsO 2 The mRNA level of Treg cytokine IL-10 in the 4-group was significantly increased, and the difference was statistically significant ( P <0.05); 25, 50 mg / L NaAsO 2 The mRNA level of Treg transcription factor Foxp3 in the 44-week group was significantly increased, and the difference was statistically significant ( P <0.05).

[0033] With 50mg / L NaAsO 2 Compared with the control group, the mRNA levels of Th1 cytokine IFN-γ and transcription factor T-bet, Th17 cytokine T-bet and transcription factor ROR-γt, Treg cytokine IL-10 and transcription factor Foxp3 in the DMF intervention group were significantly decreased, and the differences were statistically significant ( P <0.05).

[0034] 5. Changes in the expression of proteins related to the HMGB1 / TLR4 / NF-κB pathway in rats: Figure 6 As shown in B, compared with the control group, the levels of HMGB1, NF-κB p65, and TRAF6 proteins in the liver were significantly increased in the presence of 25 and 50 mg / L NaAsO 2 The difference was statistically significant ( P <0.05); 10, 50 mg / L NaAsO 2 The MyD88 protein level in the two groups was increased, and the difference was statistically significant ( P <0.05); in addition, 50 mg / L NaAsO 2 The levels of TLR4 and MD2 proteins were upregulated in the group, and the difference was statistically significant ( P <0.05). Figure 6 As shown in D, with 50mg / L NaAsO 2 Compared with the control group, the DMF intervention group down-regulated the TLR4 protein level, and the difference was statistically significant ( P <0.05).

[0035] The results are as follows Figure 7 As shown in B, compared with the control group, the levels of HMGB1 and MyD88 proteins in the kidney gradually increased with the increase of the exposure dose, and the difference was statistically significant ( P <0.05); MD2 protein level was 10, 50 mg / L NaAsO 2 The difference was statistically significant ( P <0.05); NF-κB p65 protein level was 10, 50 mg / L NaAsO 2 The difference was statistically significant ( P <0.05); TLR4 and TRAF6 protein levels were significantly higher in each NaAsO 2 The exposure group was significantly increased, and the difference was statistically significant ( P <0.05).

[0036] like Figure 7 As shown in D, with 50mg / L NaAsO 2 Compared with the control group, the DMF intervention group down-regulated the levels of TLR4, MyD88, NF-κB p65 and TRAF6 proteins, and the differences were statistically significant ( P <0.05).

[0037] Arsenic poisoning is a public health issue of global concern. In addition to causing typical skin damage, it can also cause systemic damage to multiple organs and systems and tumors

[10] . Arsenic has been shown to be immunotoxic, and studies have shown that arsenic has immunotoxic effects on immune organs such as the thymus and spleen

[11] . In addition, more and more studies have found that long-term exposure to arsenic can lead to liver damage and liver lesions such as liver fibrosis. Studies have shown that arsenic exposure can lead to diseases such as acute renal failure [12-14]. At present, there are few reports on the immunotoxic effects of arsenic on non-immune organs such as the liver and kidneys. In-depth research on the immunomodulatory effects of arsenic exposure on non-immune organs such as the liver and kidneys, as well as its effects on the immune inflammatory microenvironment and potential mechanisms of action, may provide valuable information for clarifying the toxicity and carcinogenic effects of arsenic.

[0038] Histopathology is an important basis for judging organ damage and is considered the "gold standard" for disease diagnosis. The HE staining results of the present invention showed that the arsenic exposure group had disordered liver structure and renal tubular atrophy; the liver tissue boundaries of the DMF intervention group were slightly clearer and arranged relatively neatly compared with the arsenic exposure group, and the inflammatory cell infiltration of the kidney tissue was reduced, indicating that subchronic arsenic exposure can cause pathological damage to the liver and kidneys, and DMF intervention can reduce the degree of damage to the liver and kidneys caused by arsenic exposure.

[0039] Infiltration of inflammatory cells increases the secretion of various inflammatory cytokines, thereby impairing the body's immune function. IL-1β is the first discovered early response proinflammatory cytokine

[15] . IL-6 is a proinflammatory factor mainly produced by monocytes and macrophages and is the main regulatory mediator of inflammatory responses

[16] . TNF-α is a proinflammatory cytokine mainly secreted by monocytes and macrophages

[17] . Studies by Duan et al. have shown that arsenic exposure can cause liver and kidney damage, as well as increased expression levels of IL-1β and TNF-α

[18] .

[0040] The expression levels of TNF-α, IL-6, and IL-1β in the serum of arsenic-exposed rats in the present invention were significantly increased, indicating that arsenic exposure can induce inflammatory reactions and cause immune damage to the body. Previous studies have reported that DMF can reduce the release of inflammatory factors such as IL-6, IL-1β, and TNF-α in the intestinal tissue of mice [19-21]. The results of the present invention also showed that the levels of IL-1β, IL-6, and TNF-α in the DMF intervention group were lower than those in the arsenic-exposed group, and the difference was statistically significant. This shows that DMF can reduce the secretion of inflammatory factors and inhibit the occurrence of inflammatory reactions.

[0041] In recent years, arsenic exposure-induced imbalance of T lymphocyte subsets has received increasing attention in the context of arsenic immunotoxicity. + T lymphocytes and CD8 + T lymphocytes and CD4 / CD8 ratio are considered to be the most meaningful indicators for evaluating the balance of immune regulation. + T lymphocytes are an important component of the human immune system and play an important role in regulating the body's immune response. Duan et al.

[11] found that CD4 + The proportion of T lymphocytes decreased significantly, and the CD4 / CD8 ratio continued to decrease, indicating that CD4 + T lymphocytes are the targets of early immunotoxicity caused by arsenic exposure, and CD4 + T lymphocytes are activated and differentiated into different subsets during the immune response process. According to the characteristic transcription factors and corresponding marker cytokines, CD4 +T lymphocytes can be divided into four subgroups: Th1, Th2, Th17 and Treg. The results of the present invention showed that arsenic exposure significantly upregulated the expression levels of Th1 characteristic cytokines IFN-γ and transcription factor T-bet in rat liver and kidney tissues, as well as the expression levels of Th17 characteristic cytokines IL-17 and transcription factor ROR-γt; arsenic exposure significantly downregulated the expression levels of Th2 characteristic cytokines IL-4 and transcription factor GATA-3 in liver and kidney tissues, as well as the expression levels of Treg characteristic cytokines IL-10 and transcription factor Foxp3 in liver tissues; while the expression levels of Treg characteristic cytokines IL-10 and transcription factor Foxp3 in kidney tissues of the arsenic exposure group were significantly increased, and the difference was statistically significant.

[0042] In summary, the present invention shows that subchronic arsenic exposure can lead to immune dysfunction and imbalance of Th1 / Th2 / Th17 / Treg subpopulation differentiation. The present invention also shows that DMF intervention can reverse the increased expression of IFN-γ, IL-17, T-bet, and ROR-γt, as well as the decreased expression of IL-4 and GATA3 caused by arsenic exposure, indicating that DMF can antagonize the Th1 / Th2 / Th17 / Treg balance disorder and CD4 + T cell subset differentiation disorder alleviates arsenic-induced immune inflammatory response in the liver and kidneys of rats.

[0043] At present, there are many studies on the mechanism of arsenic toxicity, mainly including arsenic-induced oxidative stress damage, inhibition of DNA repair after damage, leading to gene mutation and chromosomal aberration, affecting cell signal transduction pathways, cell cycle regulation, apoptosis and epigenetic regulation [4]. This study focuses on the key role of the interaction between TLR and DAMP in the immune regulation effect induced by arsenic. Toll-like receptors (TLRs) are considered to be sentinels of body defense and play an important role in the host's resistance to exogenous pathogens and maintenance of immune balance

[22] . TLR4 is a member of the TLRs family. As one of the most representative pattern recognition receptors, it activates the innate immune response by recognizing pathogen-associated molecular patterns (PAMPs) from bacteria and viruses and damage-associated molecular patterns (DAMPs) released by damaged cells or tissues [23, 24].

[0044] Our research team has previously found through meta-analysis that arsenic exposure can cause significant changes in proteins related to the TLR4 signaling pathway. This study shows that arsenic exposure can activate the TLR4 receptor and induce the downstream NF-κB signaling pathway through the MyD88-dependent pathway to promote inflammatory responses. TLR4 plays an important role in the innate immune response and is associated with organ damage caused by many toxic substances. This study further focused on which signals are involved in the activation of the TLR4 / NF-κB signaling pathway in the immune dysregulation induced by subchronic arsenic exposure. We focused on HMGB1, a major member of DAMPs. HMGB1, as the most widely studied DAMP, is a critical regulator of the immune-inflammatory network and cell signaling cascade

[25] . Under normal circumstances, HMGB1 is generally located in the cell nucleus. When stimulated by external stimuli, it is passively released to the extracellular space by necrotic cells or actively secreted to the extracellular space by immune cells, further binding to TLR4, initiating the MyD88-dependent pathway, and activating the NF-κB signaling pathway to promote inflammatory responses [7].

[0045] In the present invention, the levels of HMGB1, TLR4, MyD88, MD2, NF-κB p65 and TRAF6 proteins in the liver and kidney tissues of arsenic-exposed rats were significantly increased, indicating that the activation of the HMGB1 / TLR4 / NF-κB pathway may be involved in the immune damage of arsenic to the liver and kidneys. DMF intervention can significantly downregulate the expression level of TLR4 protein in the liver and kidney tissues of rats, as well as the expression levels of MyD88, NF-κB p65 and TRAF6 proteins in kidney tissues, and the differences are statistically significant.

[0046] In summary, subchronic arsenic exposure induced immune inflammatory response and immunotoxic effects in the liver and kidney, which may be related to arsenic exposure inducing increased release of HMGB1, activating the TLR4 / NF-κB inflammatory pathway, causing Th1 / Th2 / Th17 / Treg balance disorder, CD4 + The results of this study will provide useful research clues and theoretical basis for the clinical application of dimethyl fumarate to inhibit the immunotoxicity of arsenic.

[0047] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements 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. References: [1]Joel P, Michael B. Global threat of arsenic in groundwater.[J].Science (New York, NY),2020,368(6493):845-850. [2]Ferrario D, Gribaldo L, Hartung T. Arsenic exposure and immunotoxicity: a review including the possible influence of age and sex.[J].Curr Environ Health Rep, 2016, 3 (1):1-12.]. [3]Gera R, Singh V, Mitra S, et al. Arsenic exposure impels CD4commitment in thymus and suppress T cell cytokine secretion by increasingregulatory T cells[J / OL]. Sci Rep, 2017,7(1):7140. [4]Der-An T, Wei-Chang T, Huoy-Rou C. RKIP expression of liver and kidney after arsenic exposure.[J]. Environmental toxicology, 2017, 32(3):1079-1082. [5]Hanhui Z, Xiaolian L, Shuangnan Z, et al. Interplay between RAGEand TLR4 Regulates HMGB1-Induced Inflammation by Promoting Cell SurfaceExpression of RAGE and TLR4.[J]. Journal of immunology (Baltimore, Md. :1950),2020,205(3):767-775. [6]Jin H K, Hyemin K, Hyung J L, et al. Toll-like receptor 4 (TLR4):new insight immune and aging[J]. Immunity&Ageing,2023,20(1):67-67. [7]Pan L, Mengfei X, Hongbing C, et al. The effect of HMGB1 on theclinicopathological and prognostic features of cervical cancer.[J].Bioscience reports,2019,39(5):BSR20181016- BSR20181016. [8]Ilaria C, Tobias D, Edoardo G. Update on Treatment in MultipleSclerosis[J]. La Presse Médicale,2021,50(2):104068-. [9]A V M, Tamara D R, H C E, et al. Dimethyl fumarate blocks pro-inflammatory cytokine production via inhibition of TLR induced M1 and K63ubiquitin chain formation.[J]. Scientific reports,2016,6(1):31159.

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Claims

1. Application of dimethyl fumarate (DMF) in inhibiting arsenic-induced immune inflammatory response in rat liver and kidney.

2. The use according to claim 1, characterized in that: The DMF intervention inhibited the activation of HMGB1 / TLR4 / NF-κB signaling pathway, antagonized the Th1 / Th2 / Th17 / Treg balance disorder caused by sodium arsenite, and inhibited the activation of CD4 + T cell subset differentiation disorder reduces arsenic-induced immune inflammatory response in the liver and kidneys of rats.

3. The use according to claim 1, characterized in that: The DMF intervention reduces the secretion of inflammatory factors IL-1β, IL-6 and TNF-α, and inhibits the inflammatory response.

4. The use according to claim 2, characterized in that: The DMF intervention caused changes in the expression levels of Th1 characteristic cytokine IFN-γ and transcription factor T-bet, Th2 characteristic cytokine IL-4 and transcription factor GATA-3, Th17 characteristic cytokine IL-17 and transcription factor ROR-γt, and Treg characteristic cytokine IL-10 and transcription factor Foxp3.

5. The use according to claim 2, characterized in that: The DMF intervention downregulated the TLR4 protein level in liver and kidney tissues and the MyD88, NF-κB p65 and TRAF6 protein levels in the kidney.

6. The use according to any one of claims 1 to 5, characterized in that: The DMF intervention was intragastric administration of 60 mg / kg DMF once a day.

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