Application of hilpda in diagnosis and treatment of aili

Abstract

The application discloses application of Hilpda in AILI diagnosis and treatment, and provides application of Hilpda as a molecular marker in preparation of AILI diagnosis related products and application of Hilpda in preparation of AILI treatment drugs. When AILI occurs, the expression level of Hilpda is significantly increased, so that Hilpda becomes an ideal AILI diagnosis marker, and provides a convenient, fast and reliable method for clinical diagnosis of AILI. Meanwhile, Hilpda has adaptive protection on liver damage caused by AILI, and has a very good application prospect in the field of AILI treatment drugs.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an AILI diagnostic biomarker and its application in AILI treatment. Background Technology

[0002] In 2000, Denko N et al. first cloned the hypoxia-inducible gene 2 (HIG-2) from hypoxia-induced expression in human cervical epithelial cells cDNA. Hypoxia-induced expression of HIG-2 promotes neutral lipid deposition in cervical epithelial cells and stimulates cytokine expression. HIG-2 was described as a novel gene induced by hypoxia and glucose deprivation, but its function and regulatory mechanisms remain unclear. Studies have reported the detection of HIG-2 in atherosclerosis and fatty liver disease, suggesting that HIG-2 may play an important role in pathological lipid accumulation-related diseases. Further research has shown that HIG-2 is an important lipid droplet-associated protein, and it was subsequently named hypoxia-inducible lipid droplet-associated protein (Hilpda).

[0003] Hilpda is located at the lipid droplet formation site on the endoplasmic reticulum-lipid droplet interface and is expressed in cells prone to lipid droplet accumulation, such as adipocytes, hepatocytes, macrophages, and tumor cells. Besides hypoxia, fatty acids, peroxisome proliferator-activated receptor α (PPR-α), PPR-γ, and adrenergic agonists can also induce increased Hilpda expression.

[0004] Limited current research suggests that Hilpda may be an important protein involved in lipid droplet formation and the regulation of lipid metabolism. In hepatocytes, Hilpda promotes lipid accumulation by inhibiting triglyceride hydrolysis. Hilpda expression is significantly increased during 3T3-L1 adipocyte differentiation. However, unlike its role in inhibiting lipolysis in hepatocytes, interfering with or overexpressing Hilpda does not affect 3T3-L1 adipocyte differentiation, and Hilpda does not regulate lipolysis in adipocytes. In macrophages, Hilpda can promote plaque formation and the progression of atherosclerosis by controlling PGE2 production, foam cell formation, and lipid accumulation. However, the function and mechanism of action of Hilpda remain unclear.

[0005] In recent years, several studies have reported the role of Hilpda in liver diseases. The expression level of Hilpda in the liver tissue of high-fat mice is increased, and liver tissue-specific Hilpda knockout can reduce the triglyceride content and lipid droplet size of high-fat mice. Hilpda promotes the release of interleukin-10 in hepatocellular carcinoma cells, activates the signal transduction and transcription activator 3 pathway in natural killer cells (NK), inhibits the killing activity of NK cells, and promotes the recurrence and metastasis of hepatocellular carcinoma. Recent studies have shown that the expression of Hilpda in the liver of patients with non-alcoholic steatohepatitis-related hepatocellular carcinoma is up-regulated, and is closely related to poor prognosis of HCC.

[0006] However, there is no study on the role and mechanism of Hilpda in drug-induced liver injury (DILI). DILI is a liver injury caused by various types of prescription or non-prescription chemical drugs, biological agents, traditional Chinese medicines (TCM), natural medicines, health products, dietary supplements, and even excipients. It is an important drug-induced disease. At present, there is no consensus on the standard treatment of DILI in clinical practice. Most DILI patients can recover without any treatment or special measures: after stopping using the suspected drug, DILI usually recovers completely or nearly completely within a few days to weeks. However, some patients will develop chronic and severe progression, and these patients will not recover from liver function after stopping using the suspected drug, and the disease may continue or even worsen, posing a great challenge to treatment.

[0007] Acetaminophen (APAP), also known as paracetamol and N-acetylp-aminophenol, is a non-prescription antipyretic and analgesic drug. APAP is widely used worldwide, with various dosage forms (immediate and sustained-release tablets / capsules, suspensions, rectal suppositories, and intravenous infusion), easy to obtain, and is also a component of many antipyretic and analgesic drugs such as common cold drugs and opioid drugs. Generally, APAP is considered safe and effective at therapeutic doses (1-4 g / d), but improper use and overdose can cause drug-induced liver injury (AILI).

[0008] Based on the current understanding of the mechanism of APAP-induced liver toxicity, taking N-acetylcysteine to supplement GSH and eliminate active metabolites is an effective method for treating APAP-induced liver toxicity, and NAC is the only antidote approved for AILI. However, the protective effect of NAC only works in the early stage of APAP-induced liver toxicity, and the therapeutic effect of NAC decreases sharply several hours after taking an overdose of APAP. NAC no longer plays a protective role after 4 hours of excessive APAP administration in mice; the treatment window of NAC for patients is about 10 hours after taking an overdose of APAP. If effective treatment is not received within the first 10 hours of taking an overdose of APAP, severe AILI will progress to acute liver failure. In the case of ineffective medical treatment, liver transplantation is the last method for treating ALF patients. Therefore, it is an urgent need to find a new treatment for AILI / DILI.

[0009] The occurrence, progression and clinical prognosis of AILI depend on the interaction of damaging factors and protective factors. When the two are unbalanced, the damaging factors are greater than the protective factors, AILI occurs and eventually progresses to severe and difficult to reverse liver damage. At present, most of the studies on the mechanism of AILI focus on the mechanism of liver damage caused by AILI, and only a small number of studies focus on the adaptive protective mechanism of AILI. In-depth study of the protective molecular mechanism of AILI helps to more comprehensively understand the pathogenesis of AILI and provides a theoretical basis for preventing the occurrence and progression of AILI and finding potential therapeutic targets. SUMMARY

[0010] In view of the possible role of Hilpda in liver diseases, the inventors further studied its role and mechanism in AILI. It was found that the expression level of Hilpda significantly increased during the occurrence of AILI, and further studies found that the increase of Hilpda had an adaptive protective effect when AILI occurred.

[0011] Therefore, detecting the expression level of Hilpda can diagnose the occurrence of AILI. On this basis, the present application provides the use of Hilpda as a molecular marker in the preparation of AILI diagnosis related products.

[0012] Further, the present application provides an AILI diagnosis kit, which comprises Hilpda detection related reagents.

[0013] Preferably, the sample detected by the AILI diagnosis kit is serum. Alternatively, the sample detected by the AILI diagnosis kit can also be a liver biopsy tissue sample.

[0014] In another aspect, based on the adaptive protection of Hilpda in AILI, the present application provides an application of Hilpda in preparation of AILI therapeutic drugs; the therapeutic drugs can directly contain Hilpda protein, or can promote the expression of Hilpda by stimulating factors inducing high expression of Hilpda.

[0015] Further, the present application provides an AILI therapeutic drug, which comprises Hilpda protein, and further comprises a pharmaceutically acceptable carrier or excipient.

[0016] Alternatively, in another embodiment, the present application provides an AILI therapeutic drug, which comprises a Hilpda promoter, and the promoter promotes the expression of Hilpda by stimulating factors inducing high expression of Hilpda; and the drug further comprises a pharmaceutically acceptable carrier or excipient.

[0017] In summary, the present application provides an application of Hilpda in diagnosis and treatment of AILI. Specifically, the present application provides an application of Hilpda as a molecular marker in preparation of AILI diagnosis related products, and an application of Hilpda in preparation of AILI therapeutic drugs. The expression level of Hilpda is significantly increased when AILI occurs, so that Hilpda becomes an ideal AILI diagnosis marker, which provides a convenient, fast and reliable method for clinical diagnosis of AILI, and at the same time, Hilpda has adaptive protection on liver damage caused by AILI, and has a very good application prospect in the field of AILI therapeutic drugs.

[0018] The concept, specific structure and technical effects of the present application will be further described below with reference to the accompanying drawings, so as to fully understand the purposes, features and effects of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is an experimental flowchart of Example 1;

[0020] Figure 2 is a statistical diagram of Hilpda expression levels in liver tissues of the physiological saline group and four groups of AILI mice;

[0021] Figure 3 is a Hilpda protein band photo and a statistical diagram thereof in liver tissues of the physiological saline group and three groups of AILI mice;

[0022] Figure 4 is a Hilpda immunofluorescence staining result diagram in liver tissues of the physiological saline group and three groups of AILI mice;

[0023] Figure 5Figure 8 is a graph showing serum Hilpda levels in saline group and five groups of AILI mice;

[0024] Figure 6 Figure 9 is a correlation analysis of serum Hilpda levels and ALT levels in AILI mice after 24 hours;

[0025] Figure 7 Figure 10 is a correlation analysis of serum Hilpda levels and AST levels in AILI mice after 24 hours;

[0026] Figure 8 Figure 11 is a photograph showing immunohistochemical staining of Hilpda in human liver tissues of AILI patients and normal controls;

[0027] Figure 9 Figure 12 is a flow chart of Example 2;

[0028] Figure 10 Figure 13 is a comparison chart of serum ALT and AST levels in four groups of mice after expression of Hilpda;

[0029] Figure 11 Figure 14 is a graph showing liver necrosis area and statistics in four groups of mice after expression of Hilpda;

[0030] Figure 12 Figure 15 is a graph showing liver apoptosis area and statistics in four groups of mice after expression of Hilpda;

[0031] Figure 13 Figure 16 is a graph showing CYP2E1 levels in four groups of mice after expression of Hilpda;

[0032] Figure 14 Figure 17 is a graph showing GSH levels in four groups of mice after expression of Hilpda;

[0033] Figure 15 Figure 18 is a flow chart of Example 3;

[0034] Figure 16 Figure 19 is a comparison chart of survival rates in two groups of mice;

[0035] Figure 17 Figure 20 is a comparison chart of serum ALT and AST levels in two groups of mice after knockout of Hilpda;

[0036] Figure 18 Figure 21 is a graph showing liver necrosis area and statistics in two groups of mice after knockout of Hilpda;

[0037] Figure 19 Figure 22 is a graph showing liver apoptosis area and statistics in two groups of mice after knockout of Hilpda;

[0038] Figure 20is a statistical chart of CYP2E1 levels after Hilpda knockout in two groups of mice;

[0039] Figure 21 is a statistical chart of GSH levels after Hilpda knockout in two groups of mice. DETAILED DESCRIPTION

[0040] The experimental animal used in the application is a C57BL / 6J male mouse, and the Hilpda gene of the C57BL / 6J mouse is modified by FLOX through the principle of homologous recombination using the CRISPR / Cas9 technology to obtain a Hilpda gene conditional knockout mouse (Hilpda fl / fl ). The F1 generation mice are obtained by backcrossing the positive F0 generation mice and the wild type background mice for breeding and establishing lines. The Hilpda fl / fl liver cell specific Alb-Cre tool mouse is repeatedly mated and the genotype of the offspring mice is identified to obtain a liver cell specific Hilpda knockout mouse (Hilpda LKO ). All mice are purchased from Nanjing Jiequ Experimental Animal Co., Ltd., and subsequent breeding and feeding are carried out. All mice are raised in a clean level environment with constant temperature (24±2℃) and constant humidity (50±5%), 12 hours of light and dark alternation, and the mice can freely eat and drink water. All animal experiments are strictly in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and approval has been obtained from the Animal Ethics Committee of Renji Hospital, School of Medicine, Shanghai Jiao Tong University.

[0041] Example 1 Hilpda expression level is increased in AILI mice and humans

[0042] After fasting overnight, 30 SPF healthy male 8-week-old wild type C57BL / 6 mice were randomly divided into 6 groups, 5 in each group, and divided into a control group and APAP different time point (1h, 3h, 6h, 12h, 24h) intervention group. The control group: intraperitoneal injection of normal saline for 6 hours, APAP different time point intervention group: intraperitoneal injection of 300mg / kg APAP for 1 hour, 3 hours, 6 hours, 12 hours, and 24 hours. After the modeling was completed, 0.3% pentobarbital sodium solution was injected intraperitoneally to induce anesthesia. After the mice were completely anesthetized, the eyeball venous blood of all 6 groups of mice was taken for subsequent biochemical index detection; then the mice were sacrificed by cervical dislocation, and the liver tissue of the 1 hour, 3 hour, 6 hour, 12 hour groups and the control group was frozen at-80℃ for subsequent RT-PCR and Western blot experiments; the liver tissue was fixed in 4% paraformaldehyde for subsequent immunohistochemistry and immunofluorescence experiments. The experimental procedure is shown in Figure 1 .

[0043] The results show that, compared with the control group, the mRNA expression of Hilpda in the liver tissue of the AILI group was significantly increased at 6 hours and 12 hours (P<0.5, P<0.01) (see Figure 2 ). The Western blot results show that, compared with the control group, the protein expression of Hilpda in the liver tissue of the AILI group was significantly increased at 6 hours and 12 hours (see Figure 3 ). The immunofluorescence results show that, compared with the control group, the number of Hilpda positive cells in the liver tissue of the AILI group was significantly increased at 3 hours, 6 hours and 12 hours, and the increase was most significant at 12 hours; and the co-localization with the hepatocyte marker molecule ALB showed that Hilpda was mainly expressed in hepatocytes (see Figure 4 ).

[0044] The serum Hilpda level of the AILI mice gradually increased within 24 hours, and the Hilpda level at 3 hours, 6 hours, 12 hours and 24 hours was significantly increased compared with the control group (P<0.001) (see Figure 5 ), and the correlation analysis results of the Hilpda level and the ALT and AST levels of the AILI mice after 24 hours showed that the Hilpda level was significantly correlated with the ALT and AST levels, as shown in Figure 6 and 7 .

[0045] The above results show that the expression level of Hilpda in the liver tissue and serum of the AILI mice is increased, and Hilpda can be used as a diagnostic molecular marker of AILI.

[0046] Further, by performing immunohistochemical staining on the liver biopsy samples of the patients diagnosed as acute AILI and the normal control in Renji Hospital, we found that the expression level of Hilpda in the liver tissue of the AILI patients was also significantly increased (as shown in Figure 8 ).

[0047] Example 2: In vivo experiment of liver Hilpda overexpression alleviating APAP-induced liver injury

[0048] The experiment was divided into 4 groups, with 5 mice of 8 weeks old in each group, as follows:

[0049] 1) Control Hilpda fl / fl The mice were injected with 5x10 8 PFU of control Ad-Vector virus via the tail vein;

[0050] 2) Control Hilpda fl / fl The mice were injected with 5x10 8 PFU of overexpression Ad-Hilpda virus via the tail vein;

[0051] 3) Hilpda LKO 5x10 8 PFU of control Ad-Vector virus;

[0052] 4) Hilpda LKO 5x10 8 PFU of overexpression Ad-Hilpda virus;

[0053] The above four groups of mice were injected with adenovirus in the tail vein for 24 hours, then fasted for 12 hours, and then treated with intraperitoneal injection of 300 mg / kg APAP for 6 hours. After the treatment of the four groups of mice, the mice were anesthetized and sacrificed, the eyeball blood was taken and the liver tissue was collected. The liver tissue was divided into two parts: one part was frozen in-80℃ refrigerator for standby, and the other part was fixed in 4% paraformaldehyde. The blood sample was placed at room temperature for 30 minutes, then centrifuged at 3000g for 15 minutes, and the supernatant was the serum, which was transferred to a 1.5 mL EP tube and stored at-80℃ for standby. The experimental procedure is shown in Figure 9 .

[0054] The ALT and AST levels of the serum of the Hilpda overexpression mice were detected, and the results showed that when Hilpda was not overexpressed, the Hilpda LKO group of mice had significantly higher serum ALT (P<0.05) and AST (P<0.05) levels than the Hilpda fl / fl group. In the Hilpda LKO mice, the serum biochemical ALT (P<0.001) and AST (P<0.001) levels of the Hilpda overexpression group were significantly lower than those of the non-Hilpda overexpression group Figure 10 .

[0055] The histological damage after Hilpda overexpression was evaluated by liver histology. The results of H&E staining showed that when Hilpda was not overexpressed, the Hilpda LKO group of mice had significantly higher liver lobule necrosis area than the Hilpda fl / fl group of mice (P<0.001); in the Hilpda LKO mice, the liver lobule necrosis area of the Hilpda overexpression mice was significantly lower than that of the non-Hilpda overexpression group (P<0.001), and this phenomenon was also observed in the Hilpda fl / fl mice (P<0.001) Figure 11 .

[0056] The apoptosis of hepatocytes after Hilpda overexpression was evaluated by TUNEL staining. The results showed that when Hilpda was not overexpressed, the Hilpda LKOThe apoptosis area of the mice was significantly higher than that of the Hilpda fl / fl The apoptosis area of the mice was significantly lower than that of the Hilpda LKO The apoptosis area of the mice was significantly lower than that of the Hilpda fl / fl This phenomenon was also observed in the mice (P<0.001) Figure 12 ).

[0057] The Western blot was used to detect the expression levels of Hilpda and CYP2E1 proteins in the liver cells. The results showed that the expression level of Hilpda protein in the liver of the overexpression mice was significantly increased (P<0.001), while the expression level of CYP2E1 was not changed, indicating that the increase of Hilpda level did not affect the expression of CYP2E1 Figure 13 ). The GSH level in the liver tissue homogenate of the mice was further detected, and the results showed that there was no significant difference in the GSH level in the body of the Hilpda overexpression and non-overexpression mice after APAP treatment for 3 hours Figure 14 ).

[0058] Example 3: Hilpda knockout in the liver aggravates APAP-induced liver injury in vivo experiment

[0059] Ten 8-week-old Hilpda LKO mice were caged together, and ten Hilpda fl / fl control mice were caged together. After overnight fasting (18:00-6:00), the mice were divided into two groups as follows:

[0060] (1) Hilpda fl / fl mice were intraperitoneally injected with 300 mg / kg APAP for 12 hours;

[0061] (2) Hilpda LKO mice were intraperitoneally injected with 300 mg / kg APAP for 12 hours;

[0062] After the treatment, the mice were anesthetized and sacrificed, the eyeball blood was collected, and the liver tissue was collected by opening the abdominal cavity. The liver tissue was divided into two parts: one part was frozen in a-80°C refrigerator for standby, and the other part was fixed in 4% paraformaldehyde. The blood sample was placed at room temperature for 30 minutes, then centrifuged at 3000g for 15 minutes, and the serum was transferred to a new 1.5 mL EP tube and stored at-80°C for standby. The experimental procedure is shown in Figure 15 .

[0063] Twenty-seven 8-week-old male mice (fifteen Hilpda LKO mice and twelve Hilpda fl / flAfter overnight fasting, the mice were intraperitoneally injected with a lethal dose of APAP solution (750 mg / kg), and the survival of the mice was observed and recorded. A total of 12 Hilpda LKO Mice died, respectively, 1 died 6 hours after APAP treatment, 10 died 18 hours after APAP treatment, and 1 died 24 hours after APAP treatment. 5 Hilpda fl / fl Mice died, respectively, 1 died 6 hours after APAP treatment, 1 died 30 hours after APAP treatment, and 3 died 42 hours after APAP treatment. According to the death, the survival curve was drawn, and the results showed that compared with the control group of mice, the survival rate of mice with liver-specific knockout of Hilpda was significantly decreased (P = 0.0117) Figure 16 ).

[0064] Intraperitoneal injection of Hilpda LKO and Hilpda fl / fl After 300 mg / kg APAP in mice, the serum ALT and AST levels of mice were detected. The results showed that compared with Hilpda fl / fl control group of mice, the serum ALT (P < 0.01) and AST (P < 0.01) levels of Hilpda LKO mice were significantly increased Figure 17 ).

[0065] H&E staining showed that compared with Hilpda fl / fl control group of mice, the liver cell damage and necrosis of Hilpda LKO mice were more serious, which was centered on the central vein. Further statistics of necrosis area showed that compared with Hilpda fl / fl mice group, the necrosis area of liver lobule of Hilpda LKO mice group was significantly increased after APAP treatment (P < 0.001) Figure 18 ). TUNEL staining showed that the TUNEL positive staining area of Hilpda LKO mice was significantly larger than that of Hilpda fl / fl control group of mice. It is suggested that compared with Hilpda fl / fl mice, the apoptosis of Hilpda LKO mice was more significant after APAP treatment (P < 0.001) Figure 19 ).

[0066] In order to explore whether Hilpda affects the metabolism of APAP, we used Western blot to detect the expression level of CYP2E1, the key metabolic enzyme of APAP in liver tissue. The results showed that Hilpda LKOThe expression level of Hilpda protein in mice was significantly decreased (P<0.05), while the expression level of CYP2E1 remained unchanged, indicating that Hilpda knockout does not affect the expression of CYP2E1. Figure 20 As an antidote for NAPQI, a metabolite of APAP, the level of GSH is also crucial to the final APAP toxicity. To further clarify whether Hilpda affects the APAP detoxification process, the level of GSH in mouse liver tissue homogenate was measured. The results showed that, 3 hours after APAP treatment, the GSH levels in both knockout and non-knockout Hilpda mice were lower than those in the saline group; after APAP treatment, there was no significant difference in GSH levels between the Hilpda knockout and non-knockout groups. Figure 21 It is evident that Hilpda's protective effect against APAP-induced liver injury is not achieved through the mechanism of GSH clearing active metabolites.

[0067] 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 reagents for detecting the molecular marker Hilpda in the preparation of AILI diagnostic-related products.

2. The application of the reagent for detecting the molecular marker Hilpda as described in claim 1 in the preparation of AILI diagnostic-related products, characterized in that, The sample tested was serum.

3. The application of the reagent for detecting the molecular marker Hilpda as described in claim 1 in the preparation of AILI diagnostic-related products, characterized in that, The sample tested was a liver biopsy tissue sample.

4. Application of Hilpda in the preparation of AILI therapeutic drugs.

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