Application of laccer and its synthetase in ailli evaluation and treatment on the basis of nafld

Abstract

The application discloses application of LacCer or LacCer synthetase as a molecular marker in preparation of a product related to risk assessment of AILI on the basis of NAFLD, application of LacCer or LacCer synthetase as a molecular marker in preparation of a product related to prognosis assessment of AILI on the basis of NAFLD, application of LacCer synthetase as a target in preparation of a therapeutic drug for AILI on the basis of NAFLD, and application of a compound capable of promoting degradation or transformation of LacCer in preparation of a therapeutic drug for AILI on the basis of NAFLD. The application fully targets the role of LacCer in aggravation of AILI on the basis of NAFLD, treats the same, and uses the role of LacCer in risk and prognosis assessment, so that LacCer and its synthetase are excellent targets and molecular markers for drug treatment and disease assessment of AILI on the basis of NAFLD, and have a good clinical application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a biomarker for AILI risk assessment based on NAFLD, related targets in AILI treatment, and their applications. Background Technology

[0002] Drug-induced liver injury (DILI) refers to liver damage caused by various prescription or over-the-counter chemical drugs, biological agents, traditional Chinese medicines (TCMs), natural medicines, health products, dietary supplements, or their metabolites and even excipients. It is an important drug-induced disease. From an epidemiological perspective, the true incidence of DILI in the general population is often difficult to confirm. Due to differences in research methods, study populations, diagnostic criteria, and prescribing habits, the reported epidemiological data based on the general population from various countries vary considerably, and the true incidence may be higher. Studies in the United States report an annual incidence of DILI of 2.7 per 100,000 people in the general population. Studies in Iceland and France report annual incidence rates of 13.9 and 19.1 per 100,000 people, respectively. A prospective study from South Korea shows an annual incidence rate of 12 per 100,000 people in the general population. In contrast, the annual incidence rate of DILI in the general population in my country is at least 23.80 per 100,000 people, higher than all reported countries. The incidence of DILI in hospitalized patients is significantly higher than in the general population, ranging from approximately 1-6%. Among patients presenting with jaundice, DILI accounts for approximately 2-10%; acute DILI accounts for approximately 20% of hospitalized patients with acute liver injury.

[0003] Drug-induced liver injury (DILI) presents with complex clinical manifestations, causing almost all known types of acute, subacute, and chronic liver injury. Milder cases may only present with mild to moderate elevations in liver enzymes, while severe cases can lead to acute liver failure (ALF) or acute-on-chronic liver failure (ACLF), and even death. Furthermore, once DILI occurs, it usually interferes with the treatment of the primary disease, leading to disease progression or prolonged hospitalization, resulting in an extremely heavy social and economic burden. Moreover, drug hepatotoxicity is a leading cause of pre-marketing development failures, post-marketing warnings, and market withdrawals for new drugs.

[0004] The prevention and treatment of drug-induced liver injury (DILI) in my country faces a severe situation due to the following reasons: 1. my country is entering an aging society, with a large population suffering from multiple chronic diseases and requiring multiple medications for these conditions; 2. Non-standard and irrational drug use is widespread; 3. Medical personnel outside the field of hepatology are not yet familiar with the diagnosis and management of DILI; 4. Pharmaceutical companies' post-marketing risk management measures for drugs are inadequate; 5. The public generally lacks awareness of drug safety, especially regarding DILI. Therefore, DILI has become a prominent problem in my country.

[0005] Currently, there is no consensus in clinical practice regarding the standard treatment of liver dysfunction (DILI). Most DILI patients recover spontaneously without any treatment or special measures: after discontinuing the suspected drug, DILI usually recovers completely or nearly completely within days to weeks. However, some patients experience chronic and severe progression; in these patients, discontinuing the suspected drug not only fails to restore liver function, but the condition may persist or even worsen, posing a significant challenge to treatment.

[0006] Acetaminophen (APAP), also known as paracetamol and N-acetylp-aminophenol, is an over-the-counter antipyretic and analgesic. Since its introduction in 1955, APAP has been widely used worldwide. It is readily available in various dosage forms (immediate-release and sustained-release tablets / capsules, suspensions, rectal suppositories, and intravenous drops). Furthermore, APAP is a component of many antipyretic and analgesic drugs, such as common cold medicines and opioids. It is reported that over 60 million people in the United States regularly take APAP weekly. APAP is generally considered safe and effective at therapeutic doses (1-4 g / day), but improper use and overdose can lead to drug-induced liver injury (AILI). In the United States and the United Kingdom, APAP hepatotoxicity accounts for approximately 50% and 60% of all cases of acute liver failure (ALF), respectively. Furthermore, studies have shown that the mortality rate for AI-ALF patients after liver transplantation reaches 28%. In the United States, more than 78,000 emergency room visits, 33,000 hospitalizations, and approximately 500 deaths are associated with acute arterial liver injury (AILI) each year. A significant cause of APAP-related liver injury is unintentional abuse. Patients mistakenly believe that high doses of APAP can rapidly relieve pain, and some are unaware that other common antipyretic analgesics contain APAP, making them highly susceptible to simultaneously taking two to three APAP-containing medications, thus leading to adverse drug events.

[0007] Although most cases of AILI are caused by drug abuse (approximately 70%), treatment-related adverse events are also common. Previous studies have shown that dosage, age, and the time interval between intake and treatment are important risk factors for hepatotoxicity. HCV, chronic alcoholism, and chronic liver disease are also considered to be associated with increased APAP hepatotoxicity. Among chronic liver diseases, whether non-alcoholic fatty liver disease (NAFLD) exacerbates APAP hepatotoxicity has been a major focus of attention.

[0008] With significant changes in lifestyle and the increasing incidence of obesity and diabetes, NAFLD has become the most common liver disease in my country and globally. Although epidemiological data reported by different countries vary, the overall global prevalence of NAFLD in the general population is approximately 25%, reaching as high as 30%-40% in some European and American countries. This means that one in four people in the general population has NAFLD. Moreover, data from both domestic and international sources show a trend of NAFLD onset at younger ages, with a rapidly increasing prevalence among young people. The high prevalence of NAFLD presents new challenges for the prevention and treatment of acute liver injury (AILI), namely, AILI on a background of NAFLD. In fact, acute AILI events on a background of NAFLD are not uncommon in clinical practice, and related studies have been reported to explore AILI on a NAFLD background. In clinical research, several retrospective studies suggest that NAFLD / obesity may increase APAP-induced liver toxicity. Among patients hospitalized for APAP overdose, those with underlying NAFLD had a 4-7 times higher incidence of AILI than those without. In patients with morbid obesity, even therapeutic doses of APAP can significantly increase ALT and AST levels.

[0009] Therefore, in response to the increasing number of AILI events based on NAFLD in clinical practice, those skilled in the art are dedicated to developing a safe and reliable drug for the prevention and treatment of this related disease.

[0010] Lactosylceramide (LacCer) is a member of the small glycosphingolipid family. It primarily serves as a precursor to most higher homologous lipids, such as gangliosides, sulfates, fucosylated glycophospholipids, and complex neutral glycophospholipids, acting as a second messenger to transmit cellular signals. Furthermore, LacCer is a component of the plasma membrane's "lipid raft" and can act as a binding site or pattern recognition receptor for various extracellular ligands. Various external stimuli, such as platelet-derived growth factor, vascular endothelial growth factor, stress, cigarette smoke / nicotine, tumor necrosis factor-α, and especially oxidized low-density lipoprotein, can activate LacCer synthase, thereby promoting LacCer synthesis.

[0011] Limited current research suggests that LacCer primarily promotes the development of related diseases by mediating inflammatory and oxidative stress pathways. LacCer can upregulate the expression of cell adhesion molecules at the transcriptional and translational levels by activating various molecules such as cPLA2, RAS, and JNK, thereby recruiting neutrophils and monocytes for infiltration and further promoting the release of inflammatory factors such as TNF-α. This pathological process may be associated with various inflammatory diseases, such as Parkinson's disease, Alzheimer's disease, skin inflammation and alopecia, ulcerative colitis, Crohn's disease, and chronic obstructive pulmonary disease. Furthermore, LacCer may promote angiogenesis by independently / inducing PKC and cPLA2 activation, thereby promoting tumor cell proliferation and migration and participating in the pathogenesis of kidney, rectal, and liver tumors. Currently, no research has explored the role of LacCer in AILI and AILI based on NAFLD. Summary of the Invention

[0012] In response to the increasing number of acetaminophen-induced liver injury (AILI) events on the basis of NAFLD in clinical practice, the inventors have found that the levels of lactose ceramide (LacCer) and its synthase are significantly increased during the process of NAFLD aggravating AILI. Further research has found that LacCer has the effect of aggravating AILI on the basis of NAFLD.

[0013] Therefore, detecting LacCer levels can assess the risk of patients developing AILI on the basis of NAFLD. Based on this, the present invention provides the application of LacCer or LacCer synthase as a molecular marker in the preparation of products related to the risk assessment of AILI on the basis of NAFLD.

[0014] Furthermore, the present invention provides a risk assessment kit for developing AILI on the basis of NAFLD, the kit including LacCer and / or LacCer synthase detection-related reagents.

[0015] On the other hand, based on the fact that LacCer can aggravate AILI on the basis of NAFLD, this invention provides the application of LacCer or LacCer synthase as a molecular marker in the preparation of products related to the prognostic assessment of AILI on the basis of NAFLD.

[0016] Furthermore, the present invention provides an AILI prognostic assessment kit based on NAFLD, the kit comprising LacCer and / or LacCer synthase detection-related reagents.

[0017] On the other hand, this invention provides the application of LacCer synthase as a therapeutic target in the preparation of AILI therapeutic drugs based on NAFLD.

[0018] Furthermore, the therapeutic agent can inhibit LacCer synthase by directly targeting it or by reducing the expression level of its encoded mRNA.

[0019] Furthermore, the present invention provides a therapeutic agent for AILI based on NAFLD, the agent comprising a LacCer synthase inhibitor, and the agent may also comprise a pharmaceutically acceptable carrier or excipient.

[0020] Alternatively, in another embodiment, the present invention provides the use of compounds that can promote the degradation or transformation of LacCer (causing it to lose its original function) in the preparation of AILI therapeutics based on NAFLD.

[0021] Furthermore, the present invention provides a therapeutic agent for AILI based on NAFLD, the agent comprising compounds that can promote the degradation or transformation of LacCer, and the agent may also comprise a pharmaceutically acceptable carrier or excipient.

[0022] In summary, this invention provides the application of LacCer and its synthase in the assessment and treatment of AILI (Alternative Intracranial Leukemia) based on NAFLD. Specifically, it provides the application of LacCer or LacCer synthase as molecular markers in the preparation of products related to risk assessment of AILI on the basis of NAFLD; the application of LacCer or LacCer synthase as molecular markers in the preparation of products related to prognostic assessment of AILI on the basis of NAFLD; the application of LacCer synthase as a target in the preparation of therapeutic drugs for AILI on the basis of NAFLD; and the application of compounds that can promote the degradation or transformation of LacCer in the preparation of therapeutic drugs for AILI on the basis of NAFLD. LacCer and its synthase play a significant role in the assessment and treatment of AILI on the basis of NAFLD, and are excellent targets and molecular markers for drug therapy and disease assessment, with promising clinical application prospects.

[0023] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

[0024] Figure 1 This is the experimental flowchart for Example 1;

[0025] Figure 2 This is a statistical graph showing the CellTiter-Glo luminescence cell viability assay results of four groups of primary hepatocytes;

[0026] Figure 3 This is a statistical chart of CCK8 detection results for four groups of primary hepatocytes;

[0027] Figure 4 This is the experimental flowchart for Example 2;

[0028] Figure 5 This is a comparison chart of the body weights of six groups of mice after being fed a high-fat diet and a normal-fat diet;

[0029] Figure 6 This is a comparison chart of ALT levels in six groups of mice after treatment;

[0030] Figure 7 This is a comparison chart of AST levels in six groups of mice after treatment;

[0031] Figure 8 This is a comparison chart of the proportion of hepatocyte necrosis in six groups of mice after treatment;

[0032] Figure 9 These are principal component analysis diagrams of the four groups of mice in Example 3;

[0033] Figure 10 This is a volcanic eruption diagram showing the changes in sphingolipid molecule content before and after APAP treatment in normal-fat and high-fat mice;

[0034] Figure 11 This is a mountain-shaped diagram showing the changes in LacCer content before and after APAP treatment in normal-fat and high-fat mice;

[0035] Figure 12 This is a statistical graph showing the relative expression of LacCer synthase B4galt5 in the liver tissues of four groups of mice;

[0036] Figure 13 This is a statistical graph showing the relative expression of LacCer synthase B4galt6 in the liver tissues of four groups of mice;

[0037] Figure 14 This is a statistical graph showing the relative expression of LacCer synthase B4galt5 in four groups of primary hepatocytes;

[0038] Figure 15 This is a statistical graph showing the relative expression of LacCer synthase B4galt6 in four groups of primary hepatocytes;

[0039] Figure 16 This is the experimental flowchart for Example 6;

[0040] Figure 17 This is a statistical comparison chart of cell viability of four groups of primary hepatocytes after treatment;

[0041] Figure 18This is the experimental flowchart for Example 6;

[0042] Figure 19 This is a statistical graph showing the efficiency of si-B4galt5 and si-B4galt6 in interfering with LacCer synthase expression.

[0043] Figure 20 This is a statistical comparison of hepatocyte activity under conditions where the synthases B4galt5 and B4galt6 that interfere with LacCer are regulated, as well as when both are regulated.

[0044] Figure 21 This is the experimental flowchart for Example 8;

[0045] Figure 22 This is a statistical comparison chart of ALT levels in eight groups of mice;

[0046] Figure 23 This is a statistical comparison chart of AST levels in eight groups of mice;

[0047] Figure 24 This is a statistical comparison chart of the hepatocyte necrosis rate in eight groups of mice. Detailed Implementation

[0048] The experimental animals used in this invention were male C57BL / 6J mice, purchased from Nanjing Jicui Laboratory Animal Co., Ltd. All mice were induced and fed at Shanghai Southern Model Organisms Co., Ltd. All mice were housed in a clean-grade environment with constant temperature (24±2℃) and humidity (50±5%), with 12-hour light-dark cycles, and were allowed free access to food and water. All animal experiments were conducted strictly in accordance with the National Institutes of Health's guidelines for the care and use of laboratory animals, and were approved by the Animal Ethics Committee of Renji Hospital affiliated with Shanghai Jiao Tong University School of Medicine.

[0049] Example 1: In vitro experiment on NAFLD exacerbating APAP-induced liver injury

[0050] Hepatocytes were extracted from 6-8 week old wild-type (WT) male C57BL / 6J mice and seeded at equal densities in culture dishes and / or culture plates. The mice were randomly divided into 4 groups, as detailed below. Figure 1 Based on literature reports, we selected OA (oleic acid) and PA (palmitic acid) to act on adherent mouse primary hepatocytes to construct a NAFLD hepatocyte model. Specifically, OA and PA were dissolved in 1% BSA to prepare a 1 mM (0.33 mM OA + 0.66 mM PA) free fatty acid mixture, which was incubated for 24 hours. APAP was dissolved in DMEM to prepare a 10 mM APAP mixture for constructing an AILI hepatocyte model.

[0051] The CTRL group served as a normal control. After culturing in 1% BSA medium for 24 hours, the medium was replaced with DMEM, and experiments were conducted 12 hours later. The OA / PA group served as a NAFLD hepatocyte model group. After incubating with OA / PA for 24 hours, the medium was replaced with DMEM, and experiments were conducted 12 hours later. The APAP group served as an AILI hepatocyte model group. After culturing in 1% BSA for 24 hours, the medium was replaced with 10mM APAP, and experiments were conducted 12 hours later. The OA / PA+APAP group served as an AILI hepatocyte model group developed on the basis of NAFLD. After incubating with OA / PA for 24 hours, the medium was replaced with 10mM APAP, and experiments were conducted 12 hours later.

[0052] After culturing in 1% BSA medium or OA / PA for 24 hours, Oil Red O staining was performed. Compared with control primary hepatocytes, the number of lipid droplets in OA / PA-treated hepatocytes was significantly increased, indicating that the in vitro NAFLD hepatocyte model was successfully constructed.

[0053] To clarify whether steatotic hepatocytes affected the degree of hepatocyte damage in AILI, we treated mouse primary hepatocytes, which had been treated with OA / PA or 1% BSA solvent for 24 hours as controls, with 10 mM APAP for 12 hours. Then, we assessed the hepatocyte damage using several methods: 1) using CellTiter- The luminescent cell viability assay kit and the CCK8 assay kit were used to detect hepatocyte viability, such as... Figure 2 and 3 The results showed that: 10mM APAP could lead to a decrease in hepatocyte activity, indicating the successful construction of an in vitro AILI model; in steatotic hepatocytes, APAP-induced hepatocyte damage was more severe; 2) Hepatocyte survival was detected by Calcein-AM / PI double staining of live and dead cells. The results showed that after treatment with 10mM APAP, the number of dead cells increased significantly, and in steatotic hepatocytes, APAP caused even more dead cells; 3) The intracellular ROS level was detected by utilizing the characteristic that DCFH-DA can be oxidized by ROS and emit fluorescence after entering the cell. The results showed that 10mM APAP led to an increase in ROS content in hepatocytes, and this effect was more significant in steatotic hepatocytes; 4) The changes in mitochondrial membrane potential of hepatocytes in each group were detected by the JC-1 mitochondrial membrane potential detection kit. The results showed that 10mM APAP led to an increase in the intensity of JC-1 monomeric green fluorescence in hepatocytes, that is, a decrease in mitochondrial membrane potential and an increase in permeability. This effect was more significant in steatotic hepatocytes.

[0054] In summary, compared with control hepatocytes, steatotic hepatocytes exhibited more severe cell damage, ROS generation, and mitochondrial damage after stimulation with the same concentration of APAP.

[0055] Example 2: In vivo experiment on NAFLD exacerbating APAP-induced liver injury

[0056] To further validate in vivo that AILI with NAFLD predisposition results in more severe liver injury, 36 6-8 week old male WT C57BL / 6J mice were randomly divided into four groups: CTRL (simulating normal control), 150 AILI (simulating low-dose APAP intervention), 200 AILI (simulating high-dose APAP intervention), NAFLD (simulating NAFLD), NAFLD / 150 AILI (simulating low-dose APAP intervention on NAFLD basis), and NAFLD / 200 AILI (simulating high-dose APAP intervention on NAFLD basis). Six groups (n=6 per group) were used. The CTRL, 150 AILI, and 200 AILI groups were fed a normal diet, while the NAFLD, NAFLD / 150 AILI, and NAFLD / 200 AILI groups were fed a high-fat diet. After 16 weeks of feeding, the mice were fasted for 12 hours. Then, the CTRL and NAFLD groups were injected intraperitoneally with saline, the 150 AILI and NAFLD / 150 AILI groups were injected intraperitoneally with 150 mg / kg APAP, and the 200 AILI and NAFLD / 200 AILI groups were injected intraperitoneally with 200 mg / kg APAP. Six hours after the intraperitoneal injections, the mice were anesthetized and sacrificed. Blood was collected from the eyeballs and liver tissue was collected. The experimental procedure is described below. Figure 4 .

[0057] Results: The body weight of mice in the NAFLD, NAFLD / 150AILI, and NAFLD / 200AILI groups was significantly higher than that in the CTRL, 150AILI, and 200AILI groups, respectively. Figure 5 As shown in the figure. Oil Red staining results showed that in mice fed a normal-fat diet, hepatocytes were arranged in a radial cord-like pattern around the central vein, with the hepatocyte cords anastomosing to form a network; while in mice fed a high-fat diet, hepatocytes underwent fatty degeneration, were loosely arranged, and had a large number of lipid droplets or lipid vacuoles, indicating that a NAFLD animal model was successfully established.

[0058] To further validate in vivo that NAFLD exacerbates APAP-induced liver injury, we administered equal doses of APAP (150 mg / kg or 200 mg / kg) to mice fed a high-fat diet or a control normal-fat diet for 16 weeks. Six hours after treatment, the degree of liver injury was assessed in conjunction with serum ALT and AST biochemical indicators and pathological changes.

[0059] The results showed that, compared with AILI without underlying NAFLD, ALT and AST levels were significantly higher in patients with liver injury induced by the same dose of APAP on the basis of NAFLD. Figure 6 and Figure 7The area of ​​liver tissue necrosis was significantly larger. Figure 8 In cases of acute liver injury (AILI), liver damage is more severe. NAFLD can increase the severity of liver damage in AILI patients.

[0060] Example 3 Transcriptomics Sequencing

[0061] To further explore the potential mechanism by which NAFLD exacerbates AILI, we administered intraperitoneal injections of 200 mg / kg APAP solution to mice fed a normal-fat diet and mice fed a high-fat diet for 16 weeks. Six hours later, the mice were anesthetized and sacrificed, and liver tissue was collected. RNA was extracted, constructed into libraries, and then subjected to transcriptome sequencing and analysis. The results showed that the overall transcriptome changes after APAP stimulation in mice with NAFLD (NAFLD / AILI vs. NAFLD) were more significant than those in mice fed a normal-fat diet (AILI vs. CTRL). PCA analysis showed significant differentiation among the four groups (see [link to PCA analysis]). Figure 9 Differential gene analysis showed that in control mice with AILI, 388 genes were upregulated with a fold change greater than 2-fold (P < 0.05), and 385 genes were downregulated. In contrast, in mice with NAFLD, 1080 genes were upregulated with a fold change greater than 2-fold (P < 0.05), and 945 genes were downregulated, indicating that AILI on the basis of NAFLD leads to more significant transcriptomic changes. Further Venn plot analysis revealed that 699 genes were upregulated only in AILI on the basis of NAFLD, but not in AILI in control mice; and 764 genes were downregulated only in AILI on the basis of NAFLD, but not in AILI in control mice.

[0062] Next, we performed KEGG pathway cluster analysis on 699 genes upregulated when AILI occurred only on the basis of NAFLD and 764 genes downregulated when AILI occurred only on the basis of NAFLD. The results showed that the upregulated differentially regulated genes clustered in signaling pathways such as PI3K-AKT, MAPK, Rap1, sphingolipid metabolism, and TNF, while the metabolic-related pathways clustered only in the sphingolipid metabolism pathway; the downregulated differentially regulated genes clustered in metabolic pathways such as metabolic pathways, fatty acid metabolism, glucose metabolism, and steroid metabolism.

[0063] We integrated and compared the differentially regulated metabolic pathways in AILI in control mice and AILI in mice with NAFLD. We found that multiple metabolic pathways, including fatty acid metabolism, glucose metabolism, and steroid metabolism, were significantly downregulated in AILI in NAFLD-based mice, with only the sphingolipid metabolism pathway being upregulated. GSEA analysis showed that APAP stimulation did not alter the sphingolipid metabolism pathway in control mice, but in NAFLD mice, APAP stimulation significantly upregulated the sphingolipid metabolism pathway (NES = 1.58, P = 0.02).

[0064] Example 4: Lipidomics Detection

[0065] As mentioned earlier, through transcriptomics detection and analysis, we found that the sphingolipid metabolism pathway may be involved in the pathophysiological process of NAFLD aggravating AILI. To further explore the possible mediating role of sphingolipid metabolism, we injected mice fed a normal fat diet and mice fed a high fat diet for 16 weeks into the peritoneum with 200 mg / kg APAP solution. Six hours later, liver tissue was collected for non-targeted lipidomics detection based on liquid chromatography-mass spectrometry (LC-MS). We detected the content of six major classes of sphingolipids, including ceramide, phytoceramide, lactosylceramide, sphingomyelin, sphingosyl-phosphocholine, and glucoceramide. In control mice fed a lipid-rich diet, APAP stimulation resulted in a decreasing trend in the content of all six types of sphingolipids. However, in NAFLD mice, APAP stimulation showed the most significant increasing trend among the six types of sphingolipids compared to the other five. APAP stimulation significantly increased LacCer (d 14:2 / 15:0), LacCer (d 14:2 / 17:0), LacCer (d 14:2 / 17:1), LacCer (d 14:0 / 25:0), and LacCer (d 14:0 / 27:0) levels in the liver of NAFLD mice, whereas APAP did not induce this effect in control mice fed a lipid-rich diet.

[0066] Volcano plot analysis showed that in control mice fed a lipid diet, APAP-induced stress resulted in a decrease in the levels of most sphingolipid molecules; however, in NAFLD mice, APAP-induced stress significantly increased the levels of many sphingolipid molecules, especially LacCer. Figure 10 .like Figure 11 The mountain-shaped image shows that the LacCer content increased in NAFLD mice after APAP exposure.

[0067] Example 5: Detection of LacCer synthase expression level

[0068] Transcriptomic and lipidomic analyses suggest that LacCer may be involved in the pathophysiological process of NAFLD exacerbating AILI. To further explore the potential mediating role of LacCer in NAFLD-induced AILI, we first extracted RNA from the liver tissue of model mice. RT-qPCR experiments revealed that the levels of LacCer synthases B4galt5 and B4galt6 were significantly elevated during NAFLD-induced AILI, while no significant changes were observed in control mice. Figure 12 , 13 We then extracted RNA from primary hepatocytes in each group and verified this using RT-qPCR: LacCer synthases B4galt5 and B4galt6 were significantly elevated in steatotic hepatocytes after APAP stimulation, while no significant changes were observed in control hepatocytes. (See [link to RT-qPCR]). Figure 14 , 15 .

[0069] Example 6: The role of LacCer in NAFLD-induced AILI

[0070] As previously mentioned, we found that LacCer showed increased specificity in the development of autoimmune leukemia (AILI) in NAFLD. To further investigate the role of LacCer in the aggravation of AILI in NAFLD, we constructed a steatotic hepatocyte model and examined the changes in cell viability of steatotic hepatocytes undergoing AILI after exogenous LacCer addition. In the experiment to detect changes in cell viability after exogenous LacCer addition, we divided the extracted primary mouse hepatocytes into four groups. Two groups were incubated with 1 mM OA / PA for 24 hours to establish an in vitro NAFLD hepatocyte model, while the other two groups were cultured with 1% BSA as a control. Subsequently, one group of steatotic hepatocytes and the control hepatocytes were exogenously added with LacCer (LacCer dissolved in DMSO, final concentration 50 μM), and the other group was added with an equal volume of DMSO solvent. All four groups were simultaneously treated with 10 mM APAP. After 12 hours, cell viability was detected, and double staining of live and dead cells was performed. The experimental procedure is as follows: Figure 16 As shown.

[0071] Results: NAFLD hepatocytes treated with both APAP and LacCer showed significantly lower viability than those treated with APAP alone (P < 0.05), while this effect was not observed in control hepatocytes treated with APAP. (See [link to relevant documentation]). Figure 17The number of NAFLD hepatocytes that died after simultaneous treatment with APAP and LacCer was significantly higher than that of NAFLD hepatocytes treated with APAP alone, while this effect was not observed in control hepatocytes treated with APAP. This suggests that exogenously increasing LacCer levels can exacerbate the degree of AILI in steatotic hepatocytes, but has no effect on the degree of AILI in non-steatotic hepatocytes.

[0072] Example 7: The role of reducing LacCer levels in NAFLD exacerbating AILI

[0073] B4galt5 and B4galt6 are both LacCer synthases. To investigate the role of reducing LacCer levels in NAFLD-induced AILI, we used two si-RNAs, si-B4galt5 and si-B4galt6, to reduce the expression of LacCer synthase. Then, we examined the changes in cell viability of steatotic hepatocytes after APAP exposure following reduced LacCer levels. We divided primary mouse hepatocytes into eight groups: two groups were treated with si-Con as controls; two groups were treated with si-B4galt5 to interfere with LacCer synthesis; two groups were treated with si-B4galt6 to interfere with LacCer synthesis; and two groups were treated with si-B4galt5 / 6 (si-B4galt5 and si-B4galt6) to interfere with LacCer synthesis. Twenty-four hours after transfection, the cells were incubated with 1% BSA and OA / PA for 24 hours in the two groups with the same interference to simulate controls and NAFLD models, respectively. Finally, all groups were treated with equal concentrations of APAP, and the results were measured after 12 hours. The experimental procedure is as follows: Figure 18 As shown.

[0074] Results: This embodiment successfully constructed a phenotype with downregulated LacCer synthase expression. The efficiency of siRNA interference with LacCer synthase expression was as follows: Figure 19 As shown, we then simultaneously measured the cell viability of steatotic hepatocytes and control hepatocytes after exposure to the same concentration of APAP, under conditions of interference with LacCer synthases B4galt5 and B4galt6, and under conditions of both being downregulated. The results showed that, compared with si-Con, interference with LacCer synthase expression significantly reduced the AILI level in NAFLD hepatocytes (P < 0.001), while the AILI level in control hepatocytes remained unchanged. Figure 20 Furthermore, CA / PI double staining of live and dead cells further confirmed this phenotype. These results suggest that interfering with LacCer synthase expression can alleviate the damage degree of AILI in steatotic hepatocytes.

[0075] Example 8 Animal in vivo experiment

[0076] To investigate the mediating role of LacCer in NAFLD-induced AILI at the in vivo level, 52 six-week-old male C57BL / 6J mice were randomly and equally assigned to either a normal fat diet group or a high fat diet group. After 12 weeks of feeding, the 26 mice in the normal diet group were further randomly divided into four groups: the si-Con-AAV group (control group), the si-B4galt5 / 6-AAV group (simulating a control mouse model of reduced LacCer levels), the si-Con-AAV+APAP group (simulating an AILI model), and the si-B4galt5 / 6-AAV+APAP group (simulating an AILI model with reduced LacCer levels). In the si-Con-AAV group, 5 mice were injected with control adeno-associated virus via the tail vein, followed by intraperitoneal injection of saline 4 weeks later; in the si-B4galt5 / 6-AAV group, 5 mice were injected with interfering B4galt5 / 6 adeno-associated virus via the tail vein, followed by intraperitoneal injection of saline 4 weeks later; in the si-Con-AAV+APAP group, 8 mice were injected with control adeno-associated virus via the tail vein, followed by intraperitoneal injection of 200 mg / kg APAP 4 weeks later; and in the si-B4galt5 / 6-AAV+APAP group, 8 mice were injected with interfering B4galt5 / 6 adeno-associated virus via the tail vein, followed by intraperitoneal injection of 200 mg / kg APAP 4 weeks later. Six hours after the intraperitoneal injection, the mice in all four groups were anesthetized and sacrificed, and serum and liver tissue were collected.

[0077] Twenty-six mice fed a high-fat diet for 12 weeks were randomly divided into four groups: si-Con-AAV+NAFLD group (simulating NAFLD model), si-B4galt5 / 6-AAV+NAFLD group (simulating NAFLD model with reduced LacCer levels), si-Con-AAV+NAFLD / APAP group (simulating AILI model with reduced LacCer levels), and si-B4galt5 / 6-AAV+NAFLD / APAP group (simulating AILI model based on NAFLD with reduced LacCer levels). In the si-Con-AAV+NAFLD group, 5 mice received a tail vein injection of control adeno-associated virus (AAV), followed by an intraperitoneal injection of saline 4 weeks later. In the si-B4galt5 / 6-AAV+NAFLD group, 5 mice received a tail vein injection of interfering B4galt5 / 6 AAV, followed by an intraperitoneal injection of saline 4 weeks later. In the si-Con-AAV+NAFLD / APAP group, 8 mice received a tail vein injection of control AAV, followed by an intraperitoneal injection of 200 mg / kg APAP 4 weeks later. In the si-B4galt5 / 6-AAV+NAFLD / APAP group, 8 mice received a tail vein injection of interfering B4galt5 / 6 AAV, followed by an intraperitoneal injection of 200 mg / kg APAP 4 weeks later. Six hours after the intraperitoneal injection, the mice in all four groups were anesthetized and sacrificed. Serum and liver tissue were collected, and the degree of liver damage was assessed using serum ALT (alanine aminotransferase) / AST (aspartate aminotransferase) levels and pathological changes. The experimental procedure is as follows: Figure 21 As shown.

[0078] Result: As Figure 22 and 23As shown, compared with the si-Con-AAV group, the ALT and AST levels in the si-B4galt5 / 6-AAV group showed no significant changes, suggesting that interfering with LacCer synthase does not affect normal liver function. After high-fat diet intervention, the ALT and AST levels in mice injected with si-Con-AAV and mice injected with si-B4galt5 / 6-AAV showed no significant changes, suggesting that interfering with LacCer synthase does not affect the pathophysiological process of NAFLD. After 200 mg / kg APAP attack, the ALT and AST levels in both si-Con-AAV and si-B4galt5 / 6-AAV mice increased significantly, but there was no significant difference between the two groups, suggesting that interfering with LacCer synthase does not affect the pathophysiological process of AILI. Following a 200 mg / kg APAP dose, both si-Con-AAV-injected and si-B4galt5 / 6-AAV-injected NAFLD mice showed significantly increased ALT and AST levels, but the latter showed significantly lower ALT and AST levels compared to the former, suggesting that interference with LacCer synthase can improve AILI occurring on the basis of NAFLD (P < 0.05). Figure 24 The results further confirm that injection of si-B4galt5 / 6-AAV can improve hepatocyte necrosis in AILI mice on the basis of NAFLD.

[0079] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. Application of LacCer or LacCer synthase detection reagents in the preparation of products related to risk assessment of AILI in patients with NAFLD, wherein the LacCer synthase is B4galt5 and / or B4galt6.

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