Application of ion channel in hypernatreemia aggravated hepatic ischemia-reperfusion injury

Abstract

The invention belongs to the technical field of medical biology, and particularly relates to application of an ion channel in liver ischemia reperfusion injury aggravated by hypernatreemia. According to the application of the ion channel as a target for screening, preventing, relieving or / and treating the liver ischemia-reperfusion injury aggravated by the hypernatreemia, the ion channel is at least one of ENaC and NCX-1. Research finds that the hypernatremia aggravates ischemia reperfusion injury of the liver by activating an ENaC-NCX-1-NLRP3 signal channel, resulting in ROS increase, inflammation and aggravation of apoptosis, and the application of an inhibitor Amil of ENaC and an inhibitor SN-6 of NCX-1 can reduce the hypernatremia aggravated liver IRI, which brings a new therapeutic scheme for a donor of the hypernatremia, and has a broad application prospect. The improvement of the liver utilization rate of a hypernatremia donor is facilitated.

Description

Technical Field

[0001] The present invention belongs to the field of medical biotechnology, and particularly relates to the application of ion channels in liver ischemia-reperfusion injury aggravated by hypernatremia. Background Art

[0002] Liver transplantation is an effective treatment for various end-stage liver diseases, and the donor liver mainly comes from brain-dead donors. The shortage of donor livers is one of the dilemmas faced by liver transplantation. Expanding the source of donor livers has become the key, and more and more marginal donor livers are being used in organ transplantation. A marginal donor liver refers to a donor liver with a high risk of primary graft non-function or hypofunction and delayed graft loss after transplantation. A donor with a blood sodium concentration greater than 155 mmol / L is considered a marginal donor. Using the liver of a donor with hypernatremia (serum sodium concentration higher than 145 mmol / L) will lead to an increased incidence of poor liver function in the short term after transplantation, and some studies have reported that it will also increase the postoperative mortality of patients.

[0003] Liver ischemia-reperfusion injury (IRI) is an important cause of poor function of transplanted organs. When the liver is ischemic, hepatocytes lack oxygen and energy supply, resulting in abnormal metabolism and the release of damage-associated molecular patterns (DAMPs). After the blood supply to the liver resumes, the increased oxygen supply leads to an increase in the production of reactive oxygen species (ROS), further aggravating cell damage and the inflammatory response. However, the mechanism by which hypernatremia exacerbates liver ischemia-reperfusion injury has not been reported.

[0004] ENaC is a sodium ion channel that is widely distributed in multiple organs and mainly mediates the transport of sodium ions within cells. Summary of the Invention

[0005] Aiming at the problems and deficiencies existing in the prior art, the purpose of the present invention is to provide the application of ion channels in liver ischemia-reperfusion injury aggravated by hypernatremia.

[0006] Based on the above purpose, the present invention adopts the following technical solutions: The application of an ion channel as a target for screening, preventing, alleviating or / and treating liver ischemia-reperfusion injury aggravated by hypernatremia, wherein the ion channel is at least one of ENaC and NCX-1.

[0007] Preferably, the liver ischemia-reperfusion injury aggravated by hypernatremia is manifested as the levels of ALT, AST, and LDH, the area of liver necrosis, the infiltration of MPO-positive inflammatory cells, the expression levels of pro-apoptotic proteins BAX and C-Caspase 3, the mRNA expression levels of inflammatory factors IL-1β, MCP-1, and TNF-α, the number of apoptotic cells, the number of positive cells, the content of MDA, and the fluorescence intensity of ROS staining, which are increased compared with those of liver ischemia-reperfusion injury, while the expression of anti-apoptotic protein BCL-2 and the activity contents of GSH and SOD of oxygen free radicals are decreased.

[0008] Use of a drug that inhibits ion channel activation in the preparation of a drug for preventing, alleviating, or / and treating liver ischemia-reperfusion injury aggravated by hypernatremia, wherein the ion channel is at least one of ENaC and NCX-1.

[0009] Use of an ion channel-specific inhibitor in the preparation of a drug for preventing, alleviating, or / and treating liver ischemia-reperfusion injury aggravated by hypernatremia, wherein the ion channel-specific inhibitor is at least one of an ENaC-specific inhibitor and an NCX-1-specific inhibitor.

[0010] Preferably, the ENaC-specific inhibitor is Amil, and the NCX-1-specific inhibitor is SN-6.

[0011] More preferably, Amil has a negative correlation with the levels of ALT, AST, and LDH, the area of liver necrosis, the infiltration of MPO-positive inflammatory cells, the expression levels of pro-apoptotic proteins BAX, C-Caspase 3, and NLRP3, the mRNA expression levels of inflammatory factors IL-1β, MCP-1, and TNF-α, the number of apoptotic cells, the number of positive cells, the content of MDA, and the fluorescence intensity of ROS staining, and has a positive correlation with the expression level of anti-apoptotic protein BCL-2 and the activity contents of GSH and SOD of oxygen free radicals.

[0012] More preferably, SN-6 has a negative correlation with the levels of ALT, AST, and LDH, the area of liver necrosis, the infiltration of MPO-positive inflammatory cells, the expression levels of pro-apoptotic proteins BAX, C-Caspase 3, and NLRP3, the mRNA expression levels of inflammatory factors IL-1β, MCP-1, and TNF-α, the number of apoptotic cells, the number of positive cells, the content of MDA, and the fluorescence intensity of ROS staining, and has a positive correlation with the expression level of anti-apoptotic protein BCL-2 and the activity contents of GSH and SOD of oxygen free radicals.

[0013] Use of the ENaC-NCX-1-NLRP3 signaling pathway as a target for screening, preventing, alleviating, or / and treating liver ischemia-reperfusion injury aggravated by hypernatremia, wherein ENaC and NCX-1 are ion channels.

[0014] Use of a drug for inhibiting the activation of the ENaC-NCX-1-NLRP3 signaling pathway in the preparation of a drug for preventing, alleviating or / and treating liver ischemia-reperfusion injury aggravated by hypernatremia, wherein ENaC and NCX-1 are ion channels. Beneficial effects

[0015] In this application, HS animal and cell models were constructed. A 3 mol / L NaCl solution was pumped into the tail vein to establish a stable 190 mmol / L HS animal model. Usually, during the induction period, 0.7 mL / 100 g body weight of 3 mol / L NaCl solution was pumped into the tail vein first, and then 0.7 mL / h of 3 mol / L NaCl solution was pumped in during the rising period. Generally, the blood sodium concentration reached 190 mmol / L after 3 h. Finally, the pumping speed was adjusted to 0.2 mL / h during the maintenance period to maintain the blood sodium concentration. The HS cell model was cultured with 190 mmol / L DMEM for 24 h before H / R. These HS animal and cell models simulated the process of liver transplantation with hypernatremic donor livers and provided a tool for studying the role of hypernatremia in liver I / R.

[0016] This application's research found that hypernatremia exacerbates liver ischemia-reperfusion injury by activating the ENaC-NCX-1-NLRP3 signaling pathway, leading to increased ROS, aggravated inflammation and apoptosis. Application of the ENaC inhibitor Amil and the NCX-1 inhibitor SN-6 can alleviate the liver IRI aggravated by hypernatremia, which brings a new treatment plan for hypernatremic donors and is beneficial to improving the liver utilization rate of hypernatremic donors. Description of the drawings

[0017] Figure 1In it, A is the flow chart for constructing the Lewis rat model of hypernatremia I / R; B - D are the serological results of ALT (alanine aminotransferase), AST (aspartate aminotransferase), and LDH (lactate dehydrogenase) in the sham operation group (sham group), simple hypernatremia group (HS group), simple I / R group (I / R group), and hypernatremia combined with I / R group (HS + I / R group) of Lewis rats, respectively; E - F are the HE staining pictures showing the liver ischemic necrosis area in the sham group, HS group, I / R group, and HS + I / R group, respectively; G - H are the immunohistochemical pictures showing the entry of MPO - positive inflammatory cells in the sham group, HS group, I / R group, and HS + I / R group, respectively; I is the western blot results of BAX, BCL - 2, and C - Caspase3 in the sham operation group (sham group), simple hypernatremia group (HS group), simple I / R group (I / R group), and hypernatremia combined with I / R group (HS + I / R group) of Lewis rats; J - L are the expression results of inflammatory factors IL - 1β, MCP - 1, and TNF - α in the above four groups. Figure 2 In it, I is the western blot results of BAX, BCL - 2, and C - Caspase3 in the sham operation group (sham group), simple hypernatremia group (HS group), simple I / R group (I / R group), and hypernatremia combined with I / R group (HS + I / R group) of Lewis rats; J - L are the expression results of inflammatory factors IL - 1β, MCP - 1, and TNF - α in the above four groups; M - N are the TUNEL staining results of the above four groups; O - P are the DHE staining results of the above four groups; Q - S are the results of GSH activity, MDA content, and SOD activity in the above four groups. Figure 3 In it, A is the flow chart for constructing the cell model of hypernatremia combined with hypoxia - reoxygenation (HS + H / R), and B is the CCK - 8 results of BRL - 3A cells treated with 190 mmol / L HS for different times before H / R; C - D are the flow cytometry results of the normal control group (control group), hypernatremia group (HS group), hypoxia - reoxygenation group (H / R group), and hypernatremia combined with hypoxia - reoxygenation group (HS + H / R group); E is the western blot results of BAX, BCL - 2, and C - Caspase3 in the above four groups. Figure 4 In it, F - G are the ROS fluorescence staining results of the normal control group (control group), hypernatremia group (HS group), hypoxia - reoxygenation group (H / R group), and hypernatremia combined with hypoxia - reoxygenation group (HS + H / R group). Figure 5Among them, A-C are the serological results of ALT, AST, and LDH in the simple I / R group (I / R group), Lewis high-sodium combined with I / R group (HS+I / R group), Lewis rat I / R group with Amil added (Amil+I / R group), and Lewis high-sodium combined with I / R group with Amil added (Amil+HS+I / R group) of Lewis rats; D-E are the HE staining diagrams showing the liver ischemic necrosis area of the above four groups; F-G are the immunohistochemical diagrams showing the entry of MPO-positive inflammatory cells in the above four groups; Figure 6 Among them, H is the western blot results of BAX, BCL-2, and C-Caspase3 in the simple I / R group (I / R group), Lewis high-sodium combined with I / R group (HS+I / R group), Lewis rat I / R group with Amil added (Amil+I / R group), and Lewis high-sodium combined with I / R group with Amil added (Amil+HS+I / R group) of Lewis rats; I-K are the expression results of inflammatory factors IL-1β, MCP-1, and TNF-α in the above four groups; L-N are the results of GSH activity, MDA content, and SOD activity in the above four groups; O-P are the TUNEL staining results in the above four groups; Q-R are the DHE staining results in the above four groups; Figure 7 Among them, A is the CCK-8 diagram of cells after pretreating BRL-3A cells with different concentrations of Amil and then performing HS+H / R treatment, and B and D are the flow cytometry results of the hypoxic and normoxic group (H / R group), high-sodium combined with hypoxic and normoxic group (HS+H / R group), hypoxic and normoxic group after Amil pretreatment (Amil+H / R group), and high-sodium hypoxic and normoxic group after Amil pretreatment (Amil+HS+H / R group); C is the western blot results of BAX, BCL-2, and C-Caspase3 in the above four groups; Figure 8 Among them, E-F are the ROS fluorescence staining results of the hypoxic and normoxic group (H / R group), high-sodium combined with hypoxic and normoxic group (HS+H / R group), hypoxic and normoxic group after Amil pretreatment (Amil+H / R group), and high-sodium hypoxic and normoxic group after Amil pretreatment (Amil+HS+H / R group); Figure 9Among them, A - C are the serological results of ALT, AST, and LDH in Lewis rats of the simple I / R group (I / R group), Lewis high - sodium combined with I / R group (HS + I / R group), Lewis rats I / R group with SN - 6 added (SN - 6 + I / R group), and Lewis high - sodium combined with I / R group with SN - 6 added (SN - 6 + HS + I / R group); D - E are the HE staining pictures showing the liver ischemic necrosis area of the above four groups; F - G are the immunohistochemical pictures showing the entry of MPO - positive inflammatory cells of the above four groups; Figure 10 Among them, H is the western blot results of BAX, BCL - 2, and C - Caspase3 in Lewis rats of the simple I / R group (I / R group), Lewis high - sodium combined with I / R group (HS + I / R group), Lewis rats I / R group with SN - 6 added (SN - 6 + I / R group), and Lewis high - sodium combined with I / R group with SN - 6 added (SN - 6 + HS + I / R group); I - K are the expression results of inflammatory factors IL - 1β, MCP - 1, and TNF - α of the above four groups; L - N are the results of GSH activity, MDA content, and SOD activity of the above four groups; O - P are the TUNEL staining results of the above four groups; Q - R are the DHE staining results of the above four groups; Figure 11 Among them, A is the CCK - 8 picture of cells after pretreatment of BRL - 3A cells with different concentrations of SN - 6 and then HS + H / R treatment, and B is the western blot results of BAX, BCL - 2, and C - Caspase3 in the hypoxic - normoxic group (H / R group), high - sodium combined with hypoxic - normoxic group (HS + H / R group), hypoxic - normoxic group after SN - 6 pretreatment (SN - 6 + H / R group), and high - sodium hypoxic - normoxic group after SN - 6 pretreatment (SN - 6 + HS + H / R group); C - D are the flow cytometry results of the above four groups; Figure 12 Among them, E - F are the ROS fluorescence staining results of the hypoxic - normoxic group (H / R group), high - sodium combined with hypoxic - normoxic group (HS + H / R group), hypoxic - normoxic group after SN - 6 pretreatment (SN - 6 + H / R group), and high - sodium hypoxic - normoxic group after SN - 6 pretreatment (SN - 6 + HS + H / R group). Detailed implementation manners

[0018] The following details the embodiments of the present invention, and the examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to explain the present invention, and should not be construed as a limitation of the present invention.

[0019] I. Materials and methods 1. Experimental animals Male Lewis rats (6 - 8 weeks old) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The rats were housed at 25 ± 2°C, 55 ± 5% humidity, with a 12 - h light / dark cycle. All rats were allowed to acclimatize to the environment for 1 week before the experiment.

[0020] Rat experimental models 2.1 Ischemia - reperfusion (I / R) rat model: A 70% warm hepatic ischemia model was established according to the literature (DOI: 10.1002 / hep.31295). The rats were anesthetized with 1% sodium pentobarbital, and the abdominal wall was longitudinally incised. The arteries, portal veins, and bile ducts of the left and middle lobes of the rat liver were clamped with vascular clamps for 1 h, followed by 6 h of reperfusion. Then the rats were sacrificed and liver specimens were obtained.

[0021] 2.2 High - sodium concentration (HS) rat model: After the rats were anesthetized with 1% sodium pentobarbital, a high - sodium solution was pumped into the rats through a tail - vein catheter. The specific steps were as follows: First, 0.7 mL of 3 mol / L NaCl solution was rapidly pumped into each 100 - g body - weight rat for 5 min; then, 0.7 mL of 3 mol / L NaCl solution was pumped in per hour for 3 h; finally, 0.2 mL of 3 mol / L NaCl solution was pumped in per hour for 6 h.

[0022] 2.3 HS + I / R rat model: After the HS rat model was established, the infusion of the high - sodium solution was stopped, and then the I / R model was continued. During the hepatic ischemia of the rats, 0.5 mL of 20% glucose solution was orally administered intermittently and 0.2 mL of sodium pentobarbital was injected subcutaneously.

[0023] 2.4 Amil + I / R rat model: Before the I / R model was established, each 1 - kg body - weight rat was intraperitoneally injected with 1 mg Amil (HY - B0285A, MedChemExpress, USA), while the SN - 6 + I / R rat model was established in the same way, except that 1 mg of SN - 6 (HY - 107658, MedChemExpress, USA) was injected per 1 - kg body - weight.

[0024] 2.5 Amil + HS + I / R rat model: 1 mg / kg Amil was intraperitoneally injected before the HS + I / R model was established, while the SN - 6 + HS + I / R rat model was established in the same way with 1 mg / kg SN - 6.

[0025] 3. Cell culture and cell models 3.1 BRL-3A cells were cultured in Dulbecco's modified Eagle medium (DMEM, Solarbio, Beijing) supplemented with 10% fetal bovine serum in a cell incubator under normal conditions (37 °C, 5% CO2). The sodium concentration in the culture medium of the HS cell model was 190 mmol / L.

[0026] 3.2 For the H / R cell model, the medium was changed to serum-free and glucose-free DMEM, and then the cells were transferred to a modular incubator (Biospherix, Lacona, NY, USA) and placed under hypoxic conditions (1% O2, 5% CO2, and 94% N2). After 6 hours of hypoxia, the cells were transferred to a normoxic incubator (95% air + 5% CO2 mixed gas) for reoxygenation for 6 hours, and the medium was replaced with a complete medium containing 10% fetal bovine serum.

[0027] 3.3 For the HS + H / R cell model, the cells were first cultured in a medium with a sodium concentration of 190 mmol / L for several hours and then cultured under H / R conditions.

[0028] 3.4 For the Amil + H / R cell model, the cells were first cultured in a culture medium containing 10 μM Amil and then cultured under H / R conditions; while the SN-6 + H / R cell model was established with 10 μM SN-6.

[0029] 3.5 For the Amil + HS + H / R cell model, the cells were first cultured in a 190 mmol / L culture medium containing 10 μM Amil and then subjected to H / R conditions; while the SN-6 + HS + H / R cell model was established with 10 μM SN-6.

[0030] 4. Biochemical analysis Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and low-density lipoprotein cholesterol (LDH) in rat serum were detected according to a commercially available kit (Nanjing Jiancheng Bioengineering Institute).

[0031] Cell Counting Kit-8 (CCK-8) assay BRL-3A cells were seeded into 96-well plates at a cell density of 5×10 3 cells / well, subjected to different treatments, and then subjected to H / R treatment. Finally, according to the instructions of the CCK-8 kit, 10 μL of CCK-8 reagent (Boster, Wuhan) was added to each well, and after incubation at 37 °C for 1 hour, the absorbance was measured at a wavelength of 450 nm.

[0032] 6. Histology and immunohistochemical staining All liver tissues were fixed with 10% formaldehyde solution and embedded in paraffin, and then cut into 5-μm sections. The paraffin sections were stained with hematoxylin and eosin (Servicebio, Wuhan) for H&E staining, and immunostained after incubation with MPO (1:100) antibody (Servicebio, Wuhan) and secondary antibody (1:200) (Servicebio, Wuhan). Images were taken using an inverted optical microscope (Olympus, Japan).

[0033] 7. Flow cytometry analysis After discarding the culture medium, the BRL-3A cells were rinsed twice with PBS, then digested with trypsin without EDTA, and the cells were collected and centrifuged at 200 g for 5 min. The cells were resuspended in 100 μL binding buffer, 5 μL AnnexinV-FITC and 5 μL propidium iodide (Beyotime, Shanghai), incubated in the dark at room temperature for 15 min, and finally detected by flow cytometry.

[0034] 8. Western blot analysis Western blot analysis was performed according to the standard protocol to detect the protein expression levels in liver tissues and cells. Proteins were extracted with RIPA reagent (Solarbio, Beijing), and the protein concentration was quantitatively analyzed with a BCA kit (Solarbio, Beijing). The proteins were separated by 10% or 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto PVDF membranes. The PVDF membranes were incubated with specific primary antibodies overnight at 4°C, and then with secondary antibodies for 1 h at 37°C. Signals were detected using an ECL chemiluminescence reagent (NCM, Suzhou) with a ChemiDoc™ MP imaging system (BioRad, Hercules, USA).

[0035] 9. PCR analysis Total RNA of liver tissues and BRL-3A cells was extracted using Trizol reagent (Invitrogen, Carlsbad, USA). After reverse-transcribing the RNA into cDNA, quantitative RT-PCR was performed according to the instructions of SYBR qPCR Master Mix reagent (Vazyme, Nanjing). GAPDH was used as an internal reference gene, and the primers used for amplifying the target gene fragments are shown in Table 2.

[0036] Table 2 Primers and their sequences

[0037] 10. Oxidative stress analysis The liver tissue specimens were quickly frozen and fixed, cut into 10-μm sections, and stained with 10 μM dihydroethidium (DHE) fluorescent probe (Servicebio, Wuhan) and DAPI (Servicebio, Wuhan), respectively. After grinding the liver tissue, the protein concentration was measured by BCA, and then the contents of MDA, SOD, and GSH (Solarbio, Beijing) in the tissue were measured according to the instructions. After removing the culture medium and rinsing the cells twice with PBS, ROS (Biosharp, Hefei) and DAPI were added to BRL-3A cells according to the instructions, and then photographed with a fluorescence microscope (Olympus, Japan).

[0038] 11. TUNEL (Terminal deoxynucleotidyl transferase dUTP nick-endlabeling) assay The paraffin-embedded liver tissue was cut into 5-μm thick sections and stained according to the instructions of the TUNEL kit (Servicebio, Wuhan). The cell nuclei in the sections were smeared with DAPI (Servicebio, Wuhan), and then smeared with an anti-fluorescence quenching solution (Servicebio, Wuhan), and images were taken using a fluorescence microscope (Olympus, Tokyo, Japan).

[0039] 12. Statistical analysis All data were analyzed using SPSS software (version 27.0) and expressed as mean ± standard deviation. Student's t-test was used for comparison between two groups, and one-way analysis of variance (ANOVA) was used for comparison among multiple groups. P < 0.05 was considered statistically significant. Statistical significance levels were indicated by *P < 0.05, **P < 0.01, and ***P < 0.001.

[0040] II. Results and analysis 1. Effect of hypernatremia on liver IRI in Lewis rats To investigate the effect of high sodium concentration on liver I / R in Lewis rats, a hypernatremia I / R model of Lewis rats was constructed by intravenous infusion of 3 mol / L NaCl and 70% liver I / R, and the construction process of this model is as Figure 1 shown in

[0041] As Figure 1 shown in B-1D, the ALT, AST, and LDH of rats in the HS+I / R group were significantly higher than those in the I / R group. As Figure 1 shown in E-1F, the liver necrosis area of rats in the HS+I / R group was significantly larger than that in the I / R group. As Figure 1 shown in G-1H, the infiltration of MPO-positive inflammatory cells in rats in the HS+I / R group increased significantly.

[0042] As shown by Figure 2 I, the expressions of pro-apoptotic proteins BAX and C-Caspase 3 were significantly increased in the I / R group of rats treated with HS, while the expression of anti-apoptotic protein BCL-2 decreased. As shown by Figure 2 J-2L, the expressions of inflammatory factors IL-1β, MCP-1 and TNF-α were significantly increased in the HS+I / R group of rats. As shown by Figure 2 M-2N, the number of apoptotic cells in the HS+I / R group of rats was significantly higher than that in the I / R group of rats.

[0043] To detect the ROS level in tissues, DHE staining was performed in this application. As shown by Figure 2 O-2P, the number of positive cells in the HS+I / R group of rats was significantly higher than that in other groups. As shown by Figure 2 Q-2S, the activities of GSH and SOD in the liver of the HS+I / R group of rats decreased significantly, while the content of MDA increased significantly.

[0044] In summary, hypernatremia aggravated IRI in the liver of Lewis rats.

[0045] 2. Effects of hypernatremia on hepatic IRI in BRL-3A cells To study the effects of high sodium concentration on H / R of hepatocytes, a HS+H / R cell model was constructed by placing BRL-3A cells in DMEM with a concentration of 190 mmol / L for 6 h of hypoxia followed by 6 h of reperfusion. The construction process of the cell model is as shown by Figure 3 A.

[0046] As shown by Figure 3 B, pretreatment of BRL-3A cells with HS before H / R would reduce cell viability, which was most obvious at 24 h. Therefore, experiments were performed on BRL-3A cells after 24 h of HS pretreatment followed by H / R.

[0047] As shown by Figure 3 C-3D, the apoptosis rate of cells increased significantly after treatment with HS+H / R. As shown by Figure 3 E, the expressions of BAX and C-Caspase 3 in the HS+H / R group increased significantly, while the expression of BCL-2 decreased significantly.

[0048] As shown by Figure 4 F-4G, the fluorescence intensity of ROS staining in cells increased after treatment with H / R, and the fluorescence intensity of ROS staining in the HS+H / R group increased more significantly.

[0049] In summary, the above results prove that hypernatremia aggravates the injury caused by H / R in BRL-3A cells.

[0050] Effect of Amil on the exacerbation of hepatic IRI by hypernatremia in Lewis rats To investigate the role of ENaC in IRI exacerbated by HS, the specific inhibitor of ENaC, Amil, was intraperitoneally injected into the rat models of IRI and HS+IRI.

[0051] As Figure 5 shown in A-5C, the levels of ALT, AST, and LDH in the rats of the Amil+HS+I / R group were significantly lower than those in the HS+I / R group. As Figure 5 shown in D-5E, the necrosis area of the liver in the rats of the Amil+HS+I / R group was significantly lower than that in the HS+I / R group. As Figure 5 shown in F-5G, the infiltration of MPO-positive inflammatory cells in the Amil+HS+I / R group was significantly reduced.

[0052] As Figure 6 shown in H, after adding Amil, the expression levels of BAX, C-Caspase 3, and NLRP3 in the Amil+HS+I / R group decreased compared with those in the HS+I / R group, while the expression level of BCL-2 increased. As Figure 6 shown in I-6K, the mRNA expression levels of IL-1β, MCP-1, and TNF-α in the liver tissues of the Amil+HS+I / R group were significantly lower than those in the HS+I / R group. As Figure 6 shown in O-6P, the number of positive cells in the Amil+HS+I / R group was reduced compared with that in the HS+I / R group. Figure 6 shown in L-6N, after adding Amil, the activities and contents of GSH and SOD in the liver tissues of the Amil+HS+I / R group increased, while the content of MDA decreased significantly. Figure 6 shown in Q-6R, the fluorescence intensity in the Amil+HS+I / R group decreased.

[0053] In summary, Amil can reduce hepatic IRI in rats by inhibiting ENaC.

[0054] Effect of Amil on the exacerbation of hepatic IRI by hypernatremia in BRL-3A cells As Figure 7 shown in A, in BRL-3A cells treated with HS+H / R, when the concentration of Amil reached 10 μmol / L, the cell viability could be significantly improved. Therefore, 10 μmol / L was used as the concentration of Amil in cell experiments.

[0055] As Figure 7 shown in B and 7D, the apoptosis rate of cells in the Amil+HS+H / R group was significantly lower than that in the HS+H / R group. Figure 7As can be seen from C, after adding Amil, the expression levels of BAX, C-Caspase 3, and NLRP3 decreased in the Amil+HS+H / R group compared to the HS+H / R group, while the expression of BCL-2 increased.

[0056] As Figure 8 can be seen from E-8F, the fluorescence intensity of ROS staining in the Amil+HS+H / R group was significantly lower than that in the HS+H / R group. In summary, Amil can reduce the H / R injury of BRL-3A cells increased by HS by inhibiting ENaC.

[0057] Effect of NCX-1 on the exacerbation of hepatic IRI by hypernatremia in Lewis rats To investigate the role of NCX-1 in HS-exacerbated IRI, the NCX-1 specific inhibitor SN-6 was injected into rats via intraperitoneal injection.

[0058] As Figure 9 can be seen from A-9C, the levels of ALT, AST, and LDH in the serum of rats in the SN-6+HS+I / R group were significantly lower than those in the HS+I / R group. As Figure 9 can be seen from D-9E, the liver necrosis area of rats in the SN-6+HS+I / R group was significantly lower. As Figure 9 can be seen from F-9G, the infiltration of MPO-positive inflammatory cells in the SN-6+HS+I / R group was significantly lower than that in the HS+I / R group.

[0059] As Figure 10 can be seen from H, adding SN-6 can reduce the expression of BAX, C-Caspase 3, and NLRP3 in HS+I / R rats and promote the expression of BCL-2. As Figure 10 can be seen from I-10K, the mRNA expression levels of IL-1β, MCP-1, and TNF-α in the SN-6+HS+I / R group were significantly lower than those in the HS+I / R group. As Figure 10 can be seen from O-10P, SN-6 can reduce the number of positive cells in the liver tissue of HS+I / R rats. Figure 10 can be seen from L-10N, the activities of GSH and SOD in the SN-6+HS+I / R group were higher than those in the HS+I / R group, and the content of MDA was the opposite. Figure 10 can be seen from Q-10R, the staining intensity of DHE in the SN-6+HS+I / R group was lower than that in the HS+I / R group.

[0060] In summary, SN-6 can reduce hepatic IRI in Lewis rats by inhibiting NCX-1.

[0061] Effect of NCX-1 on the exacerbation of hepatic IRI by hypernatremia in BRL-3A cells As Figure 11As can be seen from A, when the concentration of SN-6 reaches 10 μmol / L, it can significantly improve the viability of BRL-3A cells treated with HS+H / R. Therefore, 10 μmol / L is used as the concentration of SN-6 in cell experiments. From Figure 11 As can be seen from B, after adding SN-6, the expression levels of BAX, C-Caspase 3, and NLRP3 decreased in the SN-6+HS+H / R group compared with the HS+H / R group, while the expression of BCL-2 increased. From Figure 11 As can be seen from C-11D, the apoptosis rate of cells in the SN-6+HS+H / R group decreased significantly.

[0062] From Figure 12 As can be seen from E-12F, the fluorescence intensity of ROS staining in the SN-6+HS+H / R group was significantly lower than that in the HS+H / R group. In summary, SN-6 can reduce the H / R injury of HS-increased BRL-3A cells by inhibiting NCX-1.

[0063] In this application, a Lewis rat I / R model of hypernatremia was constructed. The study found that liver injury was aggravated in the HS+I / R group, and the levels of ALT and AST in rat serum and the area of liver ischemia and necrosis increased. At the same time, liver inflammation increased, manifested as an increase in the fluorescence intensity of DHE staining, a decrease in the activities of GSH and SOD that can scavenge oxygen free radicals, and an increase in the content of MDA, a metabolite produced by lipid peroxidation. Hypernatremia aggravated liver IRI in Lewis rats, which was related to the activation of ENaC and NCX-1. The use of specific inhibitors could reduce the negative effects of HS on liver I / R. Similar results were also obtained in BRL-3A cell experiments. The fluorescence intensity of ROS staining in cells in the HS+H / R group increased significantly, and flow cytometry showed an increase in cell apoptosis. Western blot and qPCR results showed that the expressions of ENaC, NCX-1, and NLRP3 increased in rats in the HS+I / R group and cells in the HS+H / R group, proving that the aggravation of liver IRI by hypernatremia was related to the activation of the ENaC-NCX-1-NLRP3 signaling pathway.

[0064] Under HS conditions, the expression of ENaC increases, promoting more Na + to enter the cell, which in turn promotes ENaC-mediated ROS production. The increase in ROS production aggravates liver inflammation, increases the infiltration of MPO-positive cells, and increases the expressions of IL-1β, MCP-1, and TNF-α. After applying the ENaC inhibitor Amil, the expression of ENaC can be inhibited, reducing liver inflammation and cell apoptosis.

[0065] The expression of NCX-1 increases after hepatic I / R, and the elevation of NCX-1 is more obvious after hepatic I / R with hypernatremia, which promotes the increased expression of downstream NLRP3 and aggravates cellular inflammation and apoptosis. After the application of SN-6, the production of cellular ROS decreases, the number of MPO-positive cells in the liver tissue decreases, and the inflammatory response in the liver is reduced. The expressions of BAX and C-Caspase 3 decrease, the expression of BCL-2 increases, the fluorescence intensity of TUNEL decreases, and flow cytometry shows a reduction in cellular apoptosis, demonstrating that SN-6 can reduce the apoptosis of hepatocytes.

[0066] Hypernatremia exacerbates renal ischemia-reperfusion injury through ENaC, and the specific inhibitor of ENaC reduces the expression of ENaC and alleviates hypernatremia-induced renal ischemia-reperfusion injury. NCX-1 is an exchange channel for Na+ and Ca2+, and the expression of NCX-1 increases after myocardial ischemia-reperfusion. After the application of the specific inhibitor SN-6 of NCX-1, the myocardial ischemia-reperfusion injury can be reduced. The activation of the ENaC-NCX-1-NLRP3 signaling pathway can lead to an increase in the production of ROS and an exacerbation of the inflammatory response, which has also been reported in diseases such as cystic fibrosis and high-salt-induced hypertension. Therefore, it is inferred that the aggravation of hepatic ischemia-reperfusion injury by hypernatremia is related to the activation of the ENaC-NCX-1-NLRP3 signaling pathway.

[0067] In the present invention, the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples", etc. mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0068] Although the embodiments of the present invention have been shown and described above, it can be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those of ordinary skill in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

Claims

1. Use of an ion channel as a target for screening, preventing, alleviating or / and treating liver ischemia-reperfusion injury exacerbated by hypernatremia, wherein the ion channel is at least one of ENaC and NCX-1.

2. The application according to claim 1, characterized in that The liver ischemia-reperfusion injury exacerbated by hypernatremia is manifested as the levels of ALT, AST and LDH, the area of liver necrosis, the infiltration of MPO-positive inflammatory cells, the expression levels of pro-apoptotic proteins BAX and C-Caspase 3, the mRNA expression levels of inflammatory factors IL-1β, MCP-1 and TNF-α, the number of apoptotic cells, the number of positive cells, the content of MDA, and the fluorescence intensity of ROS staining, which are increased compared with liver ischemia-reperfusion injury, while the expression of anti-apoptotic protein BCL-2 and the activity contents of GSH and SOD of oxygen free radicals are decreased.

3. Use of a drug that inhibits ion channel activation in the preparation of a drug for preventing, alleviating or / and treating liver ischemia-reperfusion injury exacerbated by hypernatremia, wherein the ion channel is at least one of ENaC and NCX-1.

4. Use of an ion channel-specific inhibitor in the preparation of a drug for preventing, alleviating or / and treating liver ischemia-reperfusion injury exacerbated by hypernatremia, wherein the ion channel-specific inhibitor is at least one of an ENaC-specific inhibitor and an NCX-1-specific inhibitor.

5. The application according to claim 4, characterized in that, The ENaC-specific inhibitor is Amil, and the NCX-1-specific inhibitor is SN-6.

6. The application according to claim 5, wherein The Amil has a negative correlation with the levels of ALT, AST and LDH, the area of liver necrosis, the infiltration of MPO-positive inflammatory cells, the expression levels of pro-apoptotic proteins BAX, C-Caspase 3 and NLRP3, the mRNA expression levels of inflammatory factors IL-1β, MCP-1 and TNF-α, the number of apoptotic cells, the number of positive cells, the content of MDA, and the fluorescence intensity of ROS staining, and has a positive correlation with the expression level of anti-apoptotic protein BCL-2 and the activity contents of GSH and SOD of oxygen free radicals.

7. The application according to claim 5, characterized in that, The SN-6 has a negative correlation with the levels of ALT, AST and LDH, the area of liver necrosis, the infiltration of MPO-positive inflammatory cells, the expression levels of pro-apoptotic proteins BAX, C-Caspase 3 and NLRP3, the mRNA expression levels of inflammatory factors IL-1β, MCP-1 and TNF-α, the number of apoptotic cells, the number of positive cells, the content of MDA, and the fluorescence intensity of ROS staining, and has a positive correlation with the expression level of anti-apoptotic protein BCL-2 and the activity contents of GSH and SOD of oxygen free radicals.

8. Use of the ENaC-NCX-1-NLRP3 signaling pathway as a target for screening, preventing, alleviating, and / or treating liver ischemia-reperfusion injury exacerbated by hypernatremia, wherein, ENaC and NCX-1 are ion channels.

9. Use of a drug for inhibiting the activation of the ENaC-NCX-1-NLRP3 signaling pathway in the preparation of a drug for preventing, alleviating, or / and treating liver ischemia-reperfusion injury with aggravated hypernatremia, wherein, ENaC and NCX-1 are ion channels.

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