Application of serum amyloid protein 1 inhibitor in preparation of medicine for preventing and treating trichloroethylene immune renal injury

Abstract

The invention discloses application of a serum amyloid protein 1 inhibitor in preparation of a medicine for preventing and treating trichloroethylene immune renal injury, and belongs to the technical field of clinical medicine. It is found that serum SAA1 of OMDT patients and trichloroethylene-sensitized mice is remarkably increased, kidney tissue SAA1 is massively expressed, and SAA1 targeting may be a scheme with application prospects. The SAA1 is used for preventing and treating immune renal injury caused by trichloroethylene so as to resist inflammation in the early stage, promote rapid recovery of early renal functions of OMDT patients and improve prognosis.

Description

Technical Field

[0001] The invention relates to the technical field of clinical medicine, and in particular to application of a serum amyloid protein 1 inhibitor in preparing a medicine for preventing and treating trichloroethylene immune kidney damage. Background Art

[0002] As an important chemical raw material, trichloroethylene (TCE) is widely used in metal cleaning, dry cleaning of clothes, production of biopharmaceuticals, pesticides, adhesives, etc. In recent years, it has been used as a raw material for the production of green refrigerant hydrofluorocarbons. After some people are exposed to TCE, they may develop a systemic immune disease characterized by large-area skin and mucous membrane damage, often accompanied by fever, systemic lymphadenopathy, and severe liver and kidney damage, etc. This disease is called occupational medicamentosa-like dermatitis induced by trichloroethylene (OMDT). The immune system of patients with occupational medicamentosa-like dermatitis (OMDT) is overactivated, and the level of inflammatory factors in the body increases sharply, which seriously affects the patient's renal function. However, there is currently no clinical drug for the prevention and treatment of renal damage in patients with occupational medicamentosa-like dermatitis, and only glucocorticoid shock therapy is used.

[0003] High-dose glucocorticoid therapy is the main treatment for OMDT patients. The glucocorticoid use regimen varies according to the clinical phenotype of the patient. However, the initial dose of hormones used by patients with exfoliative dermatitis and bullous epidermal necrolysis is generally greater than that of patients with erythema multiforme and severe erythema multiforme. The initial dose and total amount of hormones used by patients with erythema multiforme are both lower than those of patients with severe erythema multiforme, exfoliative dermatitis, and bullous epidermal necrolysis. However, there is no difference in the initial dose maintenance time and total hormone use time of patients in the four rash types. The detailed use regimen statistics are shown in Table 1 (Wu Qifeng, Xia Lihua, Liang Weihui, et al. Analysis of glucocorticoid use in patients with occupational trichloroethylene drug eruption dermatitis [J]. Chinese Occupational Medicine, 2016, 43(06): 633-638.).

[0004] Table 1 Comparison of hormone use in OMDT patients with four types of rash [M(P 0 ~P 100 )]

[0005]

[0006] Note: Compared with the erythema multiforme group, a P<0.05.

[0007] However, this therapy has the disadvantages of long hospitalization time and many complications, which often brings great physical, psychological and economic pressure to patients and their families, which is also the main defect of this treatment plan. It is urgent to seek precise treatment methods for OMDT renal injury. Exploring the pathogenesis of OMDT and finding new targets has great occupational health significance. Summary of the invention

[0008] The purpose of the present invention is to provide the use of serum amyloid protein 1 inhibitors in the preparation of drugs for preventing and treating trichloroethylene immune renal injury. The clinical application of high-dose glucocorticoid shock treatment of OMDT has significant side effects on patients, affecting the treatment effect, and there is a lack of targeted drugs for the treatment of OMDT renal injury in the clinic. In response to this problem, the present invention improves the treatment plan and uses SAA1 inhibitors for prevention and treatment to promote the rapid recovery of early renal function in OMDT patients and improve prognosis.

[0009] To achieve the above object, the present invention provides the following solutions:

[0010] One of the technical solutions of the present invention is the use of serum amyloid protein 1 inhibitor in the preparation of drugs for preventing and treating trichloroethylene immune renal injury.

[0011] The second technical solution of the present invention is a drug for preventing and treating trichloroethylene immune renal injury, wherein the drug contains a serum amyloid protein 1 inhibitor.

[0012] The third technical solution of the present invention is the use of serum amyloid protein 1 inhibitor in the preparation of drugs for preventing and treating occupational trichloroethylene drug eruption-like dermatitis.

[0013] A fourth technical solution of the present invention is a drug for preventing and treating occupational trichloroethylene drug eruption-like dermatitis, wherein the drug contains a serum amyloid protein 1 inhibitor.

[0014] Based on the above technical solution, the present invention has the following technical effects:

[0015] The present invention found that serum SAA1 was significantly increased in OMDT patients and trichloroethylene-sensitized mice, and SAA1 was expressed in large quantities in kidney tissue. Targeting SAA1 may be a promising approach. The present invention uses SAA1 to prevent and treat trichloroethylene-induced immune renal injury, to fight inflammation at an early stage, to promote rapid recovery of early renal function in OMDT patients, and to improve prognosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0017] Figure 1 SAA1 is highly expressed in TCE immune renal injury. Among them, a is the SAA1 level in mouse peripheral blood; b is the immunohistochemical result of SAA1 in mouse kidney (magnification x1000); c is the detection of SAA1 level in human peripheral blood.

[0018] Figure 2 Figure 2 shows the relationship between SAA1 and renal injury in OMDT patients. a is the linear correlation analysis between SAA1 and Cys-c; b is the linear correlation analysis between SAA1 and urinary β2-MG; c is the correlation analysis between SAA1 and urinary VCAM-1; d is the correlation analysis between SAA1 and urinary ET-1; e is the correlation analysis between SAA1 and peripheral blood ET-1.

[0019] Figure 3 SAA1 / TLR2 regulates endothelial cell inflammatory response. Among them, a is the HE results of the kidneys of each group of mice; b is the PAS staining results of the kidneys of each group of mice; c is the Cre level of each group of mice; d is the BUN level of each group of mice; e is the release of SAA1 in HRGECs induced by TNFα combined with IFNγ; f is the expression of SAA1 protein in HRGECs induced by TNFα combined with IFNγ; g is the immunoprecipitation result of SAA1 and TLR2 in HRGECs; h is that interfering with SAA1 expression significantly downregulated the expression of ICAM1, VCAM1 and phosphorylated p65 in HRGECs; i is that interfering with SAA1 can downregulate the adhesion of HRGECs to neutrophils. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0026] The embodiment of the present invention provides the use of a serum amyloid protein 1 inhibitor in the preparation of a drug for preventing and treating trichloroethylene immune renal injury.

[0027] In some specific embodiments, the serum amyloid 1 inhibitor comprises SGA360.

[0028] Serum amyloidA1 (SAA1) is an acute phase response protein. The increase of SAA1 is associated with various kidney diseases such as renal interstitial fibrosis. After binding to TLR-like receptor 2 (TLR2), SAA1 can also activate NF-κB signaling and promote cell secretion of chemokines to recruit inflammatory cells and mediate inflammatory response. The present invention found that serum SAA1 was significantly increased in OMDT patients and trichloroethylene-sensitized mice, and SAA1 was expressed in large quantities in kidney tissue. Targeting SAA1 may be a solution with application prospects. The present invention uses SAA1 to prevent and treat trichloroethylene-induced immune renal damage, to fight inflammation at an early stage, to promote the rapid recovery of early renal function in OMDT patients, and to improve prognosis.

[0029] The embodiment of the present invention also provides a medicine for preventing and treating trichloroethylene immune renal injury, wherein the medicine comprises a serum amyloid protein 1 inhibitor.

[0030] The embodiment of the present invention also provides the use of serum amyloid protein 1 inhibitor in the preparation of a drug for preventing and treating occupational trichloroethylene drug eruption-like dermatitis.

[0031] In some specific embodiments, the serum amyloid 1 inhibitor comprises SGA360.

[0032] The embodiment of the present invention further provides a drug for preventing and treating occupational trichloroethylene drug eruption-like dermatitis, wherein the drug contains a serum amyloid protein 1 inhibitor.

[0033] Example 1

[0034] 1. Case and control population

[0035] From January 2016 to January 2020, a total of 17 OMDT patients aged 15-49 years (11 males and 6 females) were recruited from Guangdong Provincial Institute of Occupational Disease Prevention and Treatment and Shenzhen Occupational Disease Prevention and Treatment Center. Plasma from all patients and urine from 7 patients were collected upon admission. The present invention also recruited 21 control subjects, aged between 18 and 49 years.

[0036] The inclusion criteria for patients were as follows: a clear history of occupational exposure to TCE and newly diagnosed with OMDT according to GBZ 185-2006 “Diagnostic Criteria for Occupational Diseases”.

[0037] Exclusion criteria: age over 60 years old, liver and kidney diseases before exposure to TCE, mental illness, chronic diseases and other serious organic diseases.

[0038] The present invention followed the Code of Ethics of the World Medical Association (Declaration of Helsinki) for human experiments and obtained research ethics approval from the First Affiliated Hospital of Anhui Medical University, ethics approval number: 2023010. Informed consent was obtained for all population studies.

[0039] 2. Patient renal function test

[0040] Blood urea nitrogen (BUN) and creatinine (Cre) levels were measured using an automated biochemical analyzer. Plasma cystatin C (Cys-c) and urine β2-microglobulin (β2-MG) were measured using an enzyme-linked immunosorbent assay (ELISA) to assess glomerular filtration rate and tubular reabsorption function, respectively. The assays were performed according to the manufacturer's instructions.

[0041] 3. Detection of renal vascular endothelial cell damage in patients

[0042] Endothelin-1 (ET-1) in peripheral blood and urine and vascular endothelial cell adhesion molecule-1 (VCAM-1) in urine were detected by ELISA to indirectly reflect the damage of renal ECs. The detection method was carried out according to the instructions.

[0043] 4. Establishment of TCE-sensitized mouse model and neutralizing antibody intervention method

[0044] 45 pathogen-free BALB / c female mice (7-8 weeks old) were purchased from Anhui Provincial Experimental Animal Center. The mice were housed in a pathogen-free room with a 12-h / 12-h light / dark cycle and fed with normal food. The mice were randomly divided into a blank control group (5 replicates), a solvent control group (5 replicates), a TCE-treated group (20 replicates), and a TCE+SGA360-treated group (15 replicates).

[0045] Mice in the blank control group did not receive any treatment.

[0046] Before the first exposure to TCE, 2×2 cm2 of hair on the back was shaved. On the first day of modeling (the first time TCE was used), 100 μL of TCE (Freund's complete adjuvant: TCE: olive oil: acetone = 10:5:2:3) was injected subcutaneously on the back of all mice in the TCE-treated group and the TCE+SGA360-treated group. On the 4th, 7th and 10th days, 100 μL of 50% TCE (TCE: olive oil: acetone = 5:2:3) was applied to the back skin of all mice. On the 17th and 19th days, 30% TCE (TCE: olive oil: acetone = 3:2:5) was applied to the back skin of mice in the TCE-treated group and the TCE+SGA360-treated group. Before applying TCE on the 17th and 19th days, 2.0 mg / kg of SAA1 inhibitor SGA360 was injected intraperitoneally into the mice in the TCE+SGA360-treated group.

[0047] The treatment of mice in the solvent control group was basically the same as that of the TCE-treated group, but they were exposed to acetone and olive oil in the same volume ratio on days 1, 4, 7, 10, 17, and 19, without TCE.

[0048] On the 20th day, all mice were further divided into sensitized positive mice (skin reaction score ≥ 1) or sensitized negative mice (skin reaction score 0) according to skin reaction. Results The sensitization rates of the blank control group and the solvent control group were both 0. The sensitization rates of the TCE-treated group were 35%, and the sensitization rates of the TCE+SGA360-treated group were 33.3%. After the scoring, all mice were killed, and 24-hour urine, peripheral blood, kidneys or renal vascular endothelial cells were collected.

[0049] All animal experiments were performed in accordance with the ARRIVE guidelines and in accordance with the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals and applicable Animal Welfare Act. Animal experiments were approved by Anhui Medical University (Ethics Approval Number: LLSC 20230343).

[0050] 5. Detection of SAA1 in human and mouse peripheral blood

[0051] The level of SAA1 in human and mouse peripheral blood was detected by ELISA. The detection process was carried out according to the instructions.

[0052] 6. Mouse Kidney Injury Detection

[0053] Periodic acid Schiff staining (PAS staining) and hematoxylin and eosin staining (HE staining) were used to evaluate the pathological changes of the kidney. 3 μm paraffin sections were dewaxed, hydrated, stained with corresponding reagents, and the pathological changes were observed under an optical microscope.

[0054] Serum BUN, Cre, Cys-c and urine β2-MG were tested to reflect glomerular filtration and tubular reabsorption functions, respectively. The operation steps were carried out according to the instructions.

[0055] 7. Mouse Renal Vascular Endothelial Cell Injury Detection

[0056] The levels of ET-1 and VCAM-1 in urine were detected to reflect the damage of renal vascular endothelial cells. The operation steps were carried out according to the instructions.

[0057] 8. Immunohistochemistry

[0058] 3 μm thick paraffin sections were dewaxed, hydrated, and then placed in citric acid for antigen retrieval. They were then blocked with goat serum and incubated with antibodies overnight at 4°C. The next day, the sections were incubated with secondary antibodies, horseradish peroxidase, and DAB staining was used to visualize the antigen-antibody complex.

[0059] 9. Western blot

[0060] Total protein of renal ECs or human glomerular endothelial cells (HRGECs) was extracted using RIPA buffer. Protein concentration was determined using a BCA kit. Proteins were then separated by SDS-PAGE. Proteins were then transferred to a PVDF membrane. Primary and secondary antibodies were incubated after milk blocking. Enhanced chemiluminescence was used to detect the signal. The grayscale values ​​of the bands were analyzed using Image J.

[0061] 10. Cell Culture

[0062] si-NC and si-SAA1 were purchased from Shanghai GeneGene.

[0063] HRGECs were cultured in ECM containing 10% FBS, 1% endothelial cell growth supplement (ECGS), and 1% penicillin / streptomycin at 37°C with 5% CO. 2 The si-RNA was transfected using Lipofectamine 2000. The transfection steps were performed according to the instruction manual.

[0064] The cells in the negative control group (NC) were only transfected with si-NC, the cells in the TNFα+IFNγ group were only treated with TNFα and IFNγ, and the cells in the TNFα+IFNγ+si-SAA1 group were transfected with si-SAA1 and then treated with TNFα and IFNγ.

[0065] The si-RNA sequences are as follows:

[0066] SAA1: Forward,5'-CAGGUGAGGAGCACACCAATT-3';

[0067] Reverse,5'-UUGGUGUGCUCCUCACCUGTT-3'.

[0068] 11. Statistical Analysis

[0069] SPSS V.23.0 and RV.4.2.2 were used for statistical analysis. Data were expressed as mean ± standard deviation (M ± SD). Differences between groups were analyzed by one-way analysis of variance (ANOVA) and least significant difference (LSD) test. In the present invention, P < 0.05 was considered statistically significant. The relationship between SAA1 and ET-1, Cys-c, VCAM-1, and β2-MG was tested by linear regression analysis.

[0070] 12. Experimental Results

[0071] 12.1 The present invention detected the level of SAA1 in the peripheral blood of TCE-sensitized mice, and the results showed that the level of SAA1 in the peripheral blood of TCE-sensitized mice was significantly higher than that of the blank control group ( Figure 1 The results of immunohistochemistry also showed that SAA1 was abundantly expressed in the kidneys of TCE-sensitized mice ( Figure 1 b). Further testing of peripheral blood SAA1 in the population also found that peripheral blood SAA1 in ONDT patients was significantly higher than that in the control group ( Figure 1 The difference was statistically significant (P<0.05).

[0072] 12.2 The present invention focuses on detecting and analyzing the relationship between peripheral blood SAA1 and renal injury. It was found that SAA1 was not significantly correlated with blood cystatin C (Cys-c) ( Figure 2 a), but with urinary β2-microglobulin (β2-MG) ( Figure 2 b), urine vascular endothelial cell adhesion molecule 1 (VCAM-1) ( Figure 2 c), urinary endothelin-1 (ET-1) ( Figure 2 d) and peripheral blood ET-1 showed significant positive correlation ( Figure 2 Middle e). This indicates that high levels of SAA1 are closely related to the damage of renal vascular endothelial cells.

[0073] 12.3 Animal experiments have shown that injection of the SAA1 inhibitor SGA360 into mice significantly reduced the pathological damage to the glomeruli and renal tubules of the mice ( Figure 3 a, b), renal function index creatinine (Cre) ( Figure 3 c) and urea nitrogen (BUN) ( Figure 3 d) decreased significantly.

[0074] In vitro experiments verified that human renal glomerular endothelial cells (HRGECs) were treated with TNFα and IFNγ proteins to simulate the immune damage of trichloroethylene to renal vascular endothelial cells, and it was found that the supernatant SAA1 increased significantly ( Figure 3 e). When HRGECs were treated with TNFα and IFNγ, the binding of cellular SAA1 to TLR2 increased ( Figure 3 f, g), and further mediated the expression of cellular ICAM1, VCAM1 and phosphorylated p65 ( Figure 3 h), and the adhesion rate of endothelial cells to neutrophils ( Figure 3 (i) Interfering with the expression of SAA1 in HRGECs by si-RNA significantly reduced the binding of SAA1 to TLR2, inhibited the expression of ICAM1, VCAM1 and phosphorylated p65, and inhibited the adhesion of endothelial cells to neutrophils.

[0075] Through the above research, the present invention draws the following conclusions: SAA1 is expressed in large quantities in the peripheral blood of OMDT patients and TCE-sensitized mice, and SAA1 accumulates in large quantities in the kidney tissue of TCE-sensitized mice and participates in the process of renal injury. In vivo and in vitro experiments have confirmed that inhibiting the expression of SAA1 in endothelial cells can inhibit the expression of adhesion molecules in vascular endothelial cells, thereby inhibiting the adhesion of neutrophils, and reducing the binding of SAA1 to TLR2, inhibiting the activation of NF-κB, and controlling inflammation. The results show that SAA1 inhibitors are more targeted and have a significant effect on the control of renal injury in patients with trichloroethylene drug eruption-like dermatitis.

[0076] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of serum amyloid protein 1 inhibitor in the preparation of drugs for preventing and treating trichloroethylene immune renal injury.

2. The use according to claim 1, characterized in that: The serum amyloid 1 inhibitors include SGA360.

3. A drug for preventing and treating trichloroethylene immune renal injury, characterized in that: The drug comprises a serum amyloid 1 inhibitor.

4. Application of serum amyloid protein 1 inhibitors in the preparation of drugs for preventing and treating occupational trichloroethylene drug eruption-like dermatitis.

5. The use according to claim 4, characterized in that: The serum amyloid 1 inhibitors include SGA360.

6. A drug for preventing and treating occupational trichloroethylene drug eruption-like dermatitis, characterized in that: The drug comprises a serum amyloid 1 inhibitor.

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