Application of iNOS inhibitor in preparation of medicine for relieving cell injury caused by toxin combined exposure

Abstract

The invention belongs to the technical field of cytotoxicity intervention, and particularly relates to application of an iNOS inhibitor in preparation of a medicine for relieving cellular injury caused by toxin combined exposure. KK-1 cells serve as a research model, it is shown for the first time that MC-LR and NaNOS combined contamination can remarkably up-regulate the expression level of iNOS protein, and it is found through tests that Nos2 gene silencing can remarkably relieve tight junction damage and cell apoptosis caused by toxin combined exposure in the KK-1 cells; it is revealed for the first time that iNOS is a core target of toxin combined exposure induced cell tight junction damage and apoptosis, and the blank of toxic mechanism research in toxin combined exposure is filled. The application provides a new direction for a detoxification strategy of environmental toxin combined exposure, provides a theoretical basis for developing an iNOS-targeted inhibitor, and also provides a new target for etiological recognition, early diagnosis and prevention of related diseases caused by environmental toxin combined exposure.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cytotoxic intervention, and particularly relates to a drug for relieving cell damage caused by combined exposure of toxins. BACKGROUND

[0002] Under the background of accelerating modern industrialization and urbanization, environmental toxin exposure has become an important factor threatening human reproductive health. A large number of epidemiological and toxicological studies have shown that environmental toxins such as heavy metals (e.g. cadmium, lead, mercury), organic pollutants (e.g. polycyclic aromatic hydrocarbons, bisphenol A, phthalate), and atmospheric particulate matter (PM2.5) can enter the human body through multiple pathways and accumulate in the reproductive system. It is particularly noteworthy that multiple toxins often exist in the form of low-dose, long-term combined exposure, and their synergistic toxicity effect is much higher than that of a single pollutant, leading to a significantly increased risk of female ovarian function damage. The clinical manifestations include decreased ovarian reserve, sex hormone secretion disorder, ovulation disorder, and premature ovarian insufficiency (POI), which seriously threaten female reproductive health and fertility.

[0003] Microcystin and nitrite are common water pollutants, which are known to have joint toxicity to multiple organs such as liver, kidney, and intestine. Ovary is one of the common target organs of microcystin and nitrite. Both MC-LR and NaNO2 can cause ovarian function decline, such as reduction of oocytes and decrease of hormone levels, indicating that these two pollutants have the potential to induce ovarian dysfunction. However, the research on the toxic effects of combined exposure of microcystin and nitrite on female reproductive system and its mechanism is still relatively lacking. Patent 202510663224.9 discloses that FeTMPyP can be used as an intervention agent to effectively alleviate the nitration stress induced by combined exposure of MC-LR and NaNO2 in mouse ovary, improve sex hormone disorder, estrous cycle disorder, and follicle atresia, and restore the fertility of mice, providing a new way for early identification and targeted intervention of ovarian-related diseases. At present, there are obvious limitations in the prevention and treatment of ovarian toxicity caused by environmental toxins. Clinically, antioxidants or hormone replacement therapy are often used for intervention, but they can only alleviate symptoms to a certain extent and cannot specifically block the core inflammatory pathways. Moreover, long-term use of hormone replacement therapy may also pose potential risks such as breast cancer and thrombosis. The mechanism of combined exposure is not well understood, single toxin models cannot simulate real environmental exposure scenarios, and the molecular mechanisms of ovarian damage under the synergistic action of multiple toxins have not been elucidated. There is a lack of targeted therapeutic drugs, and there is no specific drug in clinical practice that can protect ovarian function and be suitable for complex exposure scenarios. Therefore, finding a new drug target and treatment strategy that can effectively prevent and treat ovarian toxicity caused by combined exposure of environmental toxins has become a key problem to be solved in the field of reproductive medicine. SUMMARY

[0004] To solve the above problems, the application provides an application of an iNOS inhibitor in preparation of a medicine for relieving cell damage caused by combined exposure of toxins.

[0005] The technical scheme of the application is as follows:

[0006] In one aspect, the application provides a reagent for relieving cell damage caused by combined exposure of toxins, wherein the reagent comprises an iNOS inhibitor.

[0007] The application explores the role of iNOS in cell damage of KK-1 caused by combined exposure of MC-LR and NaNO2 by knocking down Nos2 gene through siRNA.

[0008] Preferably, the iNOS inhibitor has a concentration of 10-50nM and an action time of 12-48h.

[0009] Preferably, the iNOS inhibitor comprises siRNA specifically targeting iNOS gene.

[0010] The siRNA is si-Nos2_1 with a sense strand sequence as shown in SEQ ID NO. 1 and an antisense strand as shown in SEQ ID NO. 2, si-Nos2_2 with a sense strand sequence as shown in SEQ ID NO. 3 and an antisense strand as shown in SEQ ID NO. 4, or si-Nos2_3 with a sense strand sequence as shown in SEQ ID NO. 5 and an antisense strand as shown in SEQ ID NO. 6.

[0011] Preferably, the combined exposure of toxins is combined exposure of MC-LR and NaNO2, and the exposure model targeting iNOS can be extended to other cell types in addition to KK-1 cells.

[0012] The specific steps are as follows:

[0013] (1) Establishing a KK-1 cell model, treating the cells with MC-LR and NaNO2 to induce cell damage;

[0014] (2) During the combined exposure of MC-LR and NaNO2, transfecting siRNA-Nos2 using siRNA gene knockdown technology to target and knock down Nos2 gene;

[0015] (3) Detecting changes in indexes related to tight junction and cell apoptosis of KK-1 cells by qPCR, Western Blot and immunofluorescence staining.

[0016] The concentration of MC-LR is 8.5μM and 17μM, and the concentration of NaNO2 is 9.5mM and 19mM.

[0017] The related indexes include apoptosis, expression levels of tight junction proteins, apoptosis-related proteins and iNOS proteins.

[0018] The tight junction proteins include Occludin, CGN, JAMC, PATJ, ZO-2 and the like; the apoptosis-related proteins include Bax, Bcl-2, Caspase-3, PARP and the like.

[0019] In the second aspect, the application protects the use of the above-mentioned reagent in the preparation of a medicine for relieving cell damage caused by combined exposure to toxins.

[0020] In the third aspect, the application protects the use of the above-mentioned reagent in the preparation of a kit for detecting cell damage caused by combined exposure to toxins.

[0021] The kit contains MC-LR, NaNO2, iNOS inhibitors, cell activity detection reagents, apoptosis detection reagents and the like, and is used for preparing a kit for relieving damage caused by combined exposure to toxins.

[0022] Preferably, the use concentration of the iNOS inhibitor in the above-mentioned reagent is 10-50 nM, and the action time is 12-48 h; the combined exposure to toxins is combined exposure to MC-LR and NaNO2.

[0023] In the fourth aspect, the application protects a pharmaceutical composition containing the above-mentioned reagent.

[0024] Preferably, the above-mentioned pharmaceutical composition is used in the preparation of a medicine for relieving cell damage caused by combined exposure to toxins.

[0025] The combined application composition containing MC-LR, NaNO2, iNOS inhibitors and the like is used for toxicity intervention research in cell or animal models.

[0026] The application provides an intervention method and application for cell damage caused by combined exposure to toxins based on iNOS knockdown, and provides a composition and a kit for the application of iNOS targeting technology in the treatment of diseases related to combined exposure to toxins.

[0027] The application has the following beneficial effects:

[0028] 1. The application first shows that combined exposure to MC-LR and NaNO2 significantly up-regulates the expression level of iNOS protein, si-RNA Nos2 can effectively knock down the expression of Nos2 gene in KK-1 cells, si-RNA Nos2 can effectively relieve the tight junction damage and cell apoptosis of KK-1 cells induced by combined exposure to MC-LR and NaNO2; iNOS is first disclosed as a core target point of tight junction damage and apoptosis of cells induced by combined exposure to MC-LR and NaNO2, and the blank of the mechanism research of toxicity in combined exposure to toxins is filled.

[0029] 2、The application research results clarify the joint toxic effect target and synergistic toxic effect path of MC-LR and NaNO2 on mouse ovaries, provide a research direction for further exploring the toxic mechanism, provide a new direction for detoxification strategies of environmental toxin combined exposure, and provide a theoretical basis for developing inhibitors (such as siRNA or small molecule drugs) targeting iNOS. The intervention method and application of the present application for toxin combined exposure to knock down iNOS can provide a new means for etiological identification, early diagnosis and prevention of diseases caused by environmental toxin combined exposure. BRIEF DESCRIPTION OF DRAWINGS

[0030] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present application, and those skilled in the art can obtain other drawings according to these drawings without creative labor.

[0031] Figure 1 For MC-LR and NaNO2 activating iNOS in mouse ovarian granulosa cells (KK-1); wherein A is the expression level of iNOS protein in KK-1 cells detected by Western Blot, and B is the quantitative analysis of the expression level of the related protein.

[0032] Figure 2 For si-RNA Nos2 can effectively knock down the expression of Nos2 in KK-1 cells; wherein A is the transfection efficiency of KK-1 cells observed under fluorescence microscope, green fluorescence indicates FAM-NC, Bar = 250 μm, B is the statistical analysis of related fluorescence intensity, and C is the influence of different si-Nos2 transfection on the expression of Nos2 in KK-1 cells detected by qPCR.

[0033] Figure 3 For si-RNA Nos2 alleviates the KK-1 cell junction damage induced by the combined exposure of MC-LR and NaNO2; wherein A is the change of iNOS and tight junction protein in KK-1 cells after Nos2 knockdown using si-Nos2 and combined exposure of MC-LR and NaNO2, and B is the quantitative analysis of the expression level of the related protein using ImageJ.

[0034] Figure 4Apoptosis induced by combined exposure of MC-LR and NaNO2 in KK-1 cells was relieved by si-RNA Nos2; A is the change of apoptosis-related proteins in KK-1 cells after knocking down Nos2 expression and combined exposure of MC-LR and NaNO2, B is the quantitative analysis of the expression level of related proteins, C is the apoptosis rate of KK-1 cells observed by TUNEL staining method, green fluorescence is the apoptosis positive cells, Bar = 200 μm, D is the statistical analysis of related fluorescence intensity. DETAILED DESCRIPTION

[0035] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0036] The experimental methods used in the following experimental examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials available from commercial channels unless otherwise specified.

[0037] The present application takes KK-1 cells as a research model, and through siRNA knockdown experiment, it is found that Nos2 gene silencing significantly relieves the tight junction damage and cell apoptosis caused by combined exposure of MC-LR and NaNO2 in KK-1 cells, revealing that iNOS-mediated tight junction damage and cell apoptosis are important reasons for the destruction of granulosa cell barrier. The research results clarify the target and synergistic toxic effect path of the combined toxic effect of microcystin and nitrite on mouse ovary, provide a research direction for further exploring the toxic mechanism, and provide a theoretical basis for developing inhibitors targeting iNOS. This will provide a new perspective for revealing the toxic mechanism of combined exposure of environmental toxins on tissue function, and also provide a new target for etiological identification, early diagnosis and prevention of related diseases caused by combined exposure of environmental toxins.

[0038] Materials and methods:

[0039] 1. Test reagents

[0040] Microcystin-LR (MC-LR, purity > 95%) was purchased from Beijing Epres Technologies Development Co., Ltd.;

[0041] Sodium nitrite (NaNO2) was purchased from Shanghai McLean Biochemical Technology Co., Ltd.;

[0042] CCK8 cell activity detection kit was purchased from Anhui Baishabiological Technology Co., Ltd.;

[0043] iNOS activity detection kit was purchased from Wuhan Elire Biotech Co., Ltd.

[0044] One-step TUNEL apoptosis detection kit was purchased from Wuhan Sivier Biotech Co., Ltd.

[0045] WB reagent was purchased from Jiangsu Kangwei Century Biotech Co., Ltd.

[0046] Other reagents were analytical grade reagents.

[0047] 2. Cell processing

[0048] KK-1 cells were gifted by Professor Xu Wentao of China Agricultural University and were grown in DMEM high glucose medium containing 10% fetal calf serum (FBS) and 1% penicillin / streptomycin mixture, and were cultured in a constant temperature incubator at 37°C, 5% CO2 and saturated humidity. Cell morphology and growth state were observed daily under an inverted microscope to ensure good cell adhesion and no contamination. Fresh medium was replaced every 2-3 days, and when the cell confluence reached 80%-90%, 0.25% trypsin digestion solution containing EDTA was used for subculture. According to the previous detection results of the research group, 1 / 2 and 1 / 4 IC 50 concentrations of MC-LR (8.5 μM, 17 μM) and NaNO2 (9.5, 19 mM) were selected for subsequent cell experiments. Factorial design was used to set up separate and combined exposure KK-1 cell study groups.

[0049] 3. Statistical analysis

[0050] Experimental data were expressed as mean ± standard deviation (SD). One-way analysis of variance (ANOVA, Birmingham, UK) was used to analyze the significance of differences between groups, and then Student-Newman-Keuls test was used. P<0.05 was considered statistically significant. SPSS21.0 (Armonk, NY, USA, 2012) was used to analyze experimental data. GraphPad Prism7 (company, La Jolla, USA) was used to process images.

[0051] Example 1: MC-LR and NaNO2 combined exposure enhances iNOS expression in KK-1 cells

[0052] (1) Western Blot detection of iNOS protein expression level in KK-1 cells:

[0053] Total protein extraction and concentration determination: The KK-1 cell sample was transferred to a 2 mL grinding tube, 1 mL of pre-cooled RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor) was added, and after grinding on ice for 30 min, ultrasonic crushing was performed every 10 min. After lysis, the sample was centrifuged for 15 min (12000 rcf, 4°C), and the supernatant was collected and transferred to a new centrifuge tube. The protein concentration was determined by BCA kit combined with multifunctional enzyme labeler. According to the measured protein concentration, all samples were adjusted to the same concentration using lysis buffer, and SDS-PAGE loading buffer was added, and boiled at 100°C for 10 min to denature the protein.

[0054] Western Blot experiment: PAGE gel rapid preparation kit was used to prepare the gel according to the standard procedure, 20 μg of protein was loaded into each gel well, and electrophoresis was carried out at a constant voltage of 60 V until the bromophenol blue entered the separation gel. After the bromophenol blue reached the bottom of the gel, the voltage was changed to 120 V. After electrophoresis, the protein on the gel was transferred to an ethanol-activated PVDF membrane by wet transfer, and the transfer conditions were 60 V and 120 min. After transfer, the PVDF membrane was immersed in TBST containing 5% skim milk powder, and blocked at room temperature for 60 min. After blocking, the primary and secondary antibodies were applied according to the antibody instructions. Finally, ECL chemiluminescence reagent was used for color development, and specific bands were captured. The gray values of the target protein and the internal reference protein GAPDH bands were analyzed by ImageJ software, and the relative expression of the target protein was calculated.

[0055] From Figure 1 It can be seen that in KK-1 cells, the expression level of iNOS protein was significantly up-regulated by MC-LR combined with NaNO2, and the difference was statistically significant compared with the control group and the single exposure group (P<0.05). These results show that MC-LR combined with NaNO2 can activate iNOS in mouse ovarian granulosa cells (KK-1).

[0056] Example 2: siRNA knockdown of Nos2 gene expression in KK-1 cells

[0057] (1) siRNA transfection

[0058] KK-1 cells were cultured normally, and after passage, the KK-1 cells were inoculated into a 6-well plate with pre-placed crawling slices, and cultured to a confluence of 40%-50%. Then, carboxyfluorescein-labeled negative control sequence (FAM-NC) was transfected into the cells at a final concentration of 20 nM using lipofectamine 3000 transfection reagent. During the transfection process, the cells were maintained in DMEM high-glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin, and cultured in a 37°C, 5% CO2 incubator. At 0, 12, and 24 h after transfection, the crawling slices were removed, and the fluorescence signal was observed by confocal microscopy under an excitation wavelength of 552 nm to analyze the transfection efficiency and determine the appropriate transfection time. Then, three groups of siRNA against Nos2 (Table 1) were used to transfect KK-1 cells under the same conditions, the cells were collected and RNA was extracted, the mRNA expression of Nos2 was detected by qPCR to screen the siRNA with the most significant down-regulation effect. Finally, the siRNA with the best knockdown effect was used for transfection during the combined exposure of MC-LR and NaNO2, and was used for subsequent experiments to explore the role of iNOS in the barrier disruption of KK-1 cells caused by the combined exposure of MC-LR and NaNO2.

[0059] Table 1 siRNA sequences used in Nos2 knockdown test

[0060]

[0061] (2) qPCR detection of mRNA expression of target genes

[0062] Total RNA extraction and cDNA synthesis: KK-1 cells were collected in 2 mL grinding tubes, and 1 mL TRIzol reagent was added to completely lyse the cells. 200 μL chloroform was added, mixed well by shaking for 15 s, and incubated at room temperature for 5 min. The mixture was centrifuged at 12000 rcf for 15 min at 4 °C. The supernatant was transferred to a new centrifuge tube, and an equal volume of isopropanol was added. After mixing gently, the mixture was incubated at room temperature for 10 min, and then centrifuged at 12000 rcf for 15 min at 4 °C to precipitate the RNA. The supernatant was discarded, and 1 mL of pre-cooled 75% ethanol was added. After centrifugation at 7500 rcf for 5 min at 4 °C, the supernatant was removed, and the RNA pellet was dried at room temperature for about 5 min until it became translucent. Then, 20 μL RNase-free water was added to dissolve the RNA. The concentration and purity of the RNA were determined using a Nanodrop 2000 ultramicro spectrophotometer, and the integrity of the RNA was verified by 1% agarose gel electrophoresis (28S:18S ≈ 2:1). Then, 1 μg of total RNA was used for reverse transcription reaction, and the genomic DNA removal and reverse transcription were performed according to the instructions of the SweScript RT II first-strand cDNA synthesis kit. The synthesized cDNA was stored at -20 °C to avoid repeated freezing and thawing.

[0063] qPCR reaction: SYBR Green fluorescent dye method combined with 384 QuantStudio 7 Flex real-time PCR system was used for qPCR reaction.

[0064] The reaction system (10 μL) contained: 2x SYBR Green Master Mix 5 μL, 0.4 μM of forward and reverse primers, respectively, 10 ng of cDNA template, and RNase-free water to make up the volume to 10 μL.

[0065] The reaction program was set as follows: pre-denaturation at 95 °C for 30 s; 40 cycles of denaturation at 95 °C for 15 s, annealing / extension at 60 °C for 30 s; and melting curve analysis stage (95 °C for 10 s, 60 °C for 5 s, and 95 °C for 15 s). The primer sequences used in the qPCR experiment are shown in Table 2. The relative expression of mRNA was calculated by 2 -ΔΔCt Each sample was set up in triplicate, and each experiment was repeated three times independently.

[0066] Table 2 Primer sequences used in qPCR experiment

[0067]

[0068] From Figure 2It can be seen that, compared with the control group, the transfection efficiency of siRNA transfection KK-1 cells for 24h can reach more than 6 times. Through qPCR experiment, it is found that siRNA-Nos2 can effectively inhibit the expression of Nos2 in KK-1 cells, and the si-Nos2_1 with the best knockdown efficiency is selected for subsequent intervention experiment.

[0069] Example 3: Knocking down Nos2 alleviates tight junction damage of KK-1 cells caused by MC-LR combined with NaNO2 exposure

[0070] (1) Western Blot detection of tight junction protein levels in KK-1 cells

[0071] Total protein extraction and concentration determination: Transfer the KK-1 cell sample to a 2 mL grinding tube, add 1 mL of pre-cooled RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor), grind on ice for 30 min, and ultrasonic crushing every 10 min. After lysis, centrifuge the sample for 15 min (12000 rcf, 4℃), collect the supernatant and transfer it to a new centrifuge tube. Determine the protein concentration by BCA kit combined with multifunctional enzyme label. According to the measured protein concentration, adjust all samples to the same concentration using lysis buffer, and add SDS-PAGE loading buffer, boil at 100℃ for 10 min to denature the protein.

[0072] Western Blot experiment: PAGE gel preparation kit was used according to the standard procedure to prepare the gel, 20 μg protein was loaded into each gel well, and electrophoresis was carried out at 60V constant voltage until bromophenol blue entered the separation gel. Change the voltage to 120V until bromophenol blue reaches the bottom of the gel. After electrophoresis, transfer the protein on the gel to ethanol-activated PVDF membrane by wet transfer method, the transfer conditions are 60V and the transfer time is 120 min. After transfer, immerse the PVDF membrane in TBST containing 5% skim milk powder, and shake at room temperature for 60 min. After blocking, according to the antibody instruction book, apply the first antibody and the second antibody. Finally, use ECL chemiluminescence reagent to develop color and capture specific bands. The gray value of the target protein and the internal reference protein GAPDH band is analyzed by ImageJ software, and the relative expression amount of the target protein is calculated.

[0073] From Figure 3It can be seen that the expression of Nos2 significantly down-regulated the enhanced iNOS level of KK-1 cells induced by MC-LR combined with NaNO2, and the relative expression level of iNOS protein in the inhibition group was more than one time lower than that in the MC-LR combined with NaNO2 exposure group, so that the relative expression level of iNOS protein in the cells after exposure basically returned to the control level. And the expression level of tight junction proteins Occludin, CGN, JAMC, PATJ and ZO-2 decreased by nearly one time and returned to the normal level of the control group, which had statistical significance compared with the MC-LR combined with NaNO2 exposure group (P<0.05). This result shows that iNOS mediates the tight junction damage of KK-1 cells induced by MC-LR combined with NaNO2 exposure, and knocking down Nos2 can alleviate the tight junction damage of KK-1 cells induced by MC-LR combined with NaNO2 exposure.

[0074] Example 4: Knocking down Nos2 alleviates the apoptosis of KK-1 cells induced by MC-LR combined with NaNO2 exposure

[0075] (1) Western Blot detection of the level of apoptosis-related proteins in KK-1 cells

[0076] Total protein extraction and concentration determination: The KK-1 cell sample was transferred to a 2 mL grinding tube, 1 mL of pre-cooled RIPA lysis buffer (containing 1% protease inhibitor and 1% phosphatase inhibitor) was added, and the sample was ground on ice for 30 min, and ultrasonic crushing was performed every 10 min. After lysis, the sample was centrifuged for 15 min (12000 rcf, 4°C), and the supernatant was collected and transferred to a new centrifuge tube. The protein concentration was determined by BCA kit combined with multifunctional enzyme labeler. According to the measured protein concentration, all samples were adjusted to the same concentration using lysis buffer, and SDS-PAGE loading buffer was added, and the protein was denatured at 100°C for 10 min.

[0077] Western Blot experiment: PAGE gel rapid preparation kit was used to prepare gels according to the standard procedure, 20 μg of protein was loaded into each gel well, and electrophoresis was performed at 60 V until bromophenol blue entered the separation gel. Then the voltage was changed to 120 V until bromophenol blue reached the bottom of the gel. After electrophoresis, the protein on the gel was transferred to an ethanol-activated PVDF membrane by wet transfer, and the transfer conditions were 60 V and 120 min. After transfer, the PVDF membrane was immersed in TBST containing 5% skim milk powder, and blocked at room temperature for 60 min. After blocking, the primary and secondary antibodies were applied according to the antibody instructions. Finally, ECL chemiluminescence reagent was used for color development, and specific bands were captured. The gray values of the target protein and the internal reference protein GAPDH bands were analyzed by ImageJ software, and the relative expression amount of the target protein was calculated.

[0078] (2) TUNEL staining to detect the apoptosis rate of KK-1 cells:

[0079] KK-1 cells were seeded in pre-prepared 6-well plates. When cell confluence reached 70%–80%, the cells were treated with MC-LR, NaNO2, and a molecular intervention agent. After treatment, the cells were fixed with 4% paraformaldehyde for 15 min. Following fixation, the cells were washed three times with PBS for 5 min each time to remove residual fixative. Subsequently, the cells were permeabilized with 0.1% Triton X-100 at room temperature for 5 min, followed by washing twice with PBS to remove the permeabilizing solution. Next, following the instructions of the one-step TUNEL apoptosis detection kit, TdT incubation buffer was prepared. 50 μL of TdT buffer was added to each slide, and the slides were incubated at 37°C in a humidified chamber in the dark for 60 min. After treatment, the slides were washed three times with PBS for 5 min each time, and the PBS solution around the sample was gently wiped away with filter paper. Finally, the cells were stained and mounted using a DAPI-containing anti-fluorescence quenching mounting medium. Apoptosis in KK-1 cells was observed using confocal microscopy (excitation wavelength 488 nm), and TUNEL-positive cells were counted and statistically analyzed using ImageJ software. Each experiment was performed in triplicate.

[0080] Depend on Figure 4 Western blotting results showed that, compared with the M2N2 group, knockdown of Nos2 reduced the apoptosis-related Bax / Bcl-2 ratio by more than half, essentially restoring the Bax / Bcl-2 ratio in cells after exposure to control levels. Simultaneously, the enhanced levels of Cleaved Caspase-3 and Cleaved PARP induced by MC-LR combined with NaNO2 exposure were significantly inhibited, with their relative expression levels decreasing by more than 0.5-fold, a statistically significant difference compared to the MC-LR combined with NaNO2 exposure group (P<0.05). TUNEL staining results indicated that Nos2 knockdown significantly inhibited MC-LR combined with NaNO2 exposure-induced apoptosis in KK-1 cells, with the apoptosis rate of KK-1 cells decreasing by approximately 4-fold compared to the MC-LR combined with NaNO2 exposure group (P<0.05). This result indicates that iNOS mediates MC-LR and NaNO2 co-exposure-induced apoptosis in KK-1 cells, and knocking down Nos2 can alleviate MC-LR and NaNO2 co-exposure-induced apoptosis in KK-1 cells.

[0081] In summary, MC-LR combined with NaNO2 significantly up-regulated the expression level of iNOS protein, siRNA-Nos2 can effectively inhibit the expression of Nos2 in KK-1 cells, and knockdown of Nos2 can alleviate the tight junction damage and apoptosis of KK-1 cells caused by MC-LR combined with NaNO2. Therefore, iNOS is the core target of tight junction damage and apoptosis induced by MC-LR combined with NaNO2, and the intervention method and application of toxin combined exposure targeting iNOS knockdown can cause cell damage, which can provide a new means for the etiological identification, early diagnosis and prevention of related diseases caused by environmental toxin combined exposure.

[0082] The above merely describes preferred embodiments of the present application and is not intended to limit the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. An agent that alleviates cell damage caused by combined exposure to toxins, characterized in that: The reagent comprises an iNOS inhibitor.

2. The agent of claim 1, wherein: The iNOS inhibitor has an effective concentration of 10-50 nM and an effective time of 12-48 h.

3. The agent of claim 2, wherein: The iNOS inhibitors include siRNAs that specifically target iNOS genes.

4. The agent of claim 3, wherein: The siRNA is siRNA with a sense strand sequence as shown in SEQ ID NO. 1 and an antisense strand as shown in SEQ ID NO. 2, Nos2_1 siRNA with a sense strand sequence as shown in SEQ ID NO. 3 and an antisense strand as shown in SEQ ID NO. 4, Nos2_2 or siRNA with a sense strand sequence as shown in SEQ ID NO. 5 and an antisense strand as shown in SEQ ID NO. 6, Nos2_3 .

5. The agent according to any one of claims 1 to 4, characterized in that: The toxin combination exposure is MC-LR combined with NaNO2 exposure.

6. Use of the reagent of claim 5 in the preparation of a medicament for alleviating cell damage caused by toxin combination exposure.

7. Use of the reagent of claim 5 in the preparation of a kit for detecting cell damage caused by toxin combination exposure.

8. Use according to claim 6 or 7, characterized in that: The iNOS inhibitor has an effective concentration of 10-50 nM and an effective time of 12-48 h; and the toxin combination exposure is MC-LR combined with NaNO2 exposure.

9. A pharmaceutical composition, characterized by: The reagent comprises an iNOS inhibitor.

10. Use of the pharmaceutical composition of claim 9 in the preparation of a medicament for alleviating cell damage caused by toxin combination exposure.

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