CN112675309A - Application of DUSP12 in liver ischemia-reperfusion injury - Google Patents

Abstract

The invention discloses application of DUSP12 in liver ischemia-reperfusion injury. According to the invention, a liver cell specific DUSP12 gene knockout mouse and a liver cell specific DUSP12 transgenic mouse are selected as test objects, a mouse liver IRI injury model is respectively constructed, then separation culture of primary liver cells and construction of liver cell in vitro hypoxia/reoxygenation are carried out, and the function of DUSP12 in IRI injury is researched; the new function of DUSP12 gene in liver I/R injury is discovered, and the over-expression of DUSP12 can reduce the liver ischemia reperfusion injury by inhibiting inflammation and apoptosis, so that DUSP12 can be applied to screening or preparing the medicine for preventing/treating the liver ischemia reperfusion injury, and a new medicine target point is provided for the liver I/R injury.

Description

Application of DUSP12 in liver ischemia-reperfusion injury

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to application of DUSP12 in liver ischemia-reperfusion injury.

Background

Hepatic Ischemia Reperfusion Injury (IRI) is a common physiological phenomenon in trauma, shock and liver surgery, and severe IRI injury caused by prolonged warm ischemia often leads to liver function damage and even irreversible liver failure. Many drugs and genetic engineering methods are designed to ameliorate ischemia reperfusion injury, however, there are no clinically feasible drugs and methods to prevent and treat hepatic ischemia reperfusion injury. More and more studies have shown that the initiation of the inflammatory response of the liver is a critical step in the ischemia reperfusion injury of the liver. During liver ischemia, hypoxia and glycogen depletion lead to hepatocyte damage. Hepatocytes produce reactive oxygen species under ischemic stimulation, leading to apoptosis of hepatocytes after liver reperfusion. Subsequently, the hepatocytes recruit various inflammatory cells and induce the secretion of inflammatory mediators by apoptosis or release of necrosis factors, such as danger pattern-associated molecules (DAMPs) like high-rate mobility group protein-1 (HMGB1), Heat Shock Proteins (HSPs), etc. All of these inflammatory cells and cytokines trigger an inflammatory cascade that ultimately leads to positive feedback hepatocyte necrosis. Therefore, the specific mechanism of liver ischemia-reperfusion inflammation is clarified, and the design of targeted drugs and methods is crucial.

Dual specificity phosphatases (DUSPs) are a family of protein phosphatases that dephosphorylate threonine and tyrosine residues in the Thr-X-Tyr (TXY motif) activation motifs of mitogen-activated protein kinases (MAPKs). Some DUSPs have been found to be involved in the regulation of liver IRI damage. For example, DUSP14 protected against hepatic ischemia reperfusion injury via the DUSP14-TAK1-JNK1/2 pathway. DUSP12 is a member of the atypical DUSP protein tyrosine phosphatase family. Although it does not contain the typical MAPK targeting motif, it is still able to dephosphorylate tyrosine and serine/threonine residues. In addition to the catalytic domain, DUSP12 contains a zinc finger domain, making it one of the largest DUSPs that exhibit strong nuclear expression in several tissues.

Previous studies have shown that DUSP12 plays an important role in brown adipocyte differentiation, microbial infection, and myocardial hypertrophy. DUSP12 is also involved in stress-induced cell death and cancer. However, the pathophysiological role of DUSP12 in liver ischemia reperfusion has not been fully demonstrated.

Disclosure of Invention

The invention aims to provide an application of DUSP12 in liver ischemia-reperfusion injury, determine the relation between the expression of DUSP12 gene and the liver ischemia-reperfusion injury, and disclose a new function of DUSP12 in the liver ischemia-reperfusion injury.

In order to achieve the purpose, the invention adopts the following technical scheme:

according to the invention, a liver cell specific DUSP12 gene knockout mouse (CKO) and a liver cell specific DUSP12 transgenic mouse (TG) are selected as test objects, a mouse liver IRI injury model is respectively constructed by taking a CKO newborn mouse (Flox) and a TG newborn mouse (NTG) as controls, and then separation culture of primary liver cells and construction of liver cell in vitro hypoxia/reoxygenation (H/R) are carried out to research the function of DUSP12 in IRI injury.

The results of detection and analysis such as liver function determination, HE staining, TUNEL staining, immunohistochemistry, immunofluorescence, WB, qRT-PCR, statistical analysis and the like show that:

1. the necrotic area was significantly increased in the CKO group compared to the Flox group, liver I/R induced down-regulation of DUSP12 expression, while deficiency of DUSP12 aggravated liver inflammation during liver I/R injury and liver I/R, and aggravated apoptosis after live liver I/R.

In vivo inflammation and apoptosis of liver I/R after overexpression of DUSP12, which indicates that DUSP12 has a protective effect on liver I/R injury.

DUSP12 reduced hepatic I/R injury by inhibiting the activation of ASK1-JNK/p38 signals, which may be achieved by ASK 1-dependent mechanisms.

The invention provides application of DUSP12 in preparation of a medicine for preventing/treating hepatic ischemia-reperfusion injury.

According to the above protocol, the drug is one that is capable of promoting expression of DUSP 12.

The invention has the beneficial effects that:

the invention discovers a new function of DUSP12 gene in liver I/R injury, and DUSP12 overexpression can reduce liver ischemia reperfusion injury by inhibiting inflammation and apoptosis, so DUSP12 can be applied to screening or preparing medicines for preventing/treating liver ischemia reperfusion injury, and a new medicine target is provided for liver I/R injury.

Drawings

Figure 1 is a graph of down-regulation of expression of DUSP12 in a liver IRI model, wherein liver IRI injury is exacerbated by a deficiency of DUSP12 in hepatocytes, wherein:

(A) sham and ischemic 1h reperfusion 6h wild type mouse liver DUSP12mRNA analysis (n-4);

(B) western blot to detect the levels of liver DUSP12 protein (n-6) in sham operated and 6h post-ischemic reperfusion mice;

(C) detecting protein expression (n is 3) of primary hepatocytes DUSP12 cultured in a sham treatment group and in 6H of H/R stimulation by Western blot;

(D) 6h after IRI, typical HE staining images of ischemic liver lobes and necrotic area quantification (n ═ 6) are performed on each group of mice;

(E) sham and post-ischemic reperfusion 6h in Flox and DUSP12-CKO mice serum ALT, AST levels (n-8-9).

GADPH serves as an internal reference. All data are expressed as mean ± SD. P <0.01, N.S, not significant.

FIG. 2 is a graph of inflammatory response following deficiency of DUSP12 in induction of liver IRI; wherein:

(A) TNF- α, IL-1 β, IL-6 and CCl-2mRNA expression levels (n ═ 4) after 6h of ischemia reperfusion in FLOX and DUSP12-CKO groups;

(B) each group of mice was immuno-fluorescent stained for CD11b positive inflammatory cells (red) (n-5);

(C) immunoblot analysis of activation of hepatic NF- κ B signal after 6h of IRI stimulation in FLOX or DUSP12-CKO mice (n ═ 3).

GADPH serves as an internal reference. All data are expressed as mean ± SD. P < 0.05; p < 0.01. N.s., no significant difference.

Figure 3 shows that DUSP12 deficiency enhances apoptosis in liver IRI; wherein:

(A) typical images of reperfusion 6h liver sections and TUNEL staining quantification (20 ×), (n ═ 5);

(B) expression levels of liver Bad, Bax and Bcl-2 mrnas 6h after ischemia reperfusion in Flox and CKO mice (n ═ 4);

(C) western blot analysis of Flox and DUSP12-CKO mice IRI or Bax and Bcl-2 expression in liver 6h after sham surgery and quantitative results (n is 3);

(D) images of C-Caspase3 immunohistochemical staining of Flox and CKO mouse liver 6h after sham surgery or ischemia reperfusion (n-4).

GADPH serves as an internal reference. All data are expressed as mean ± SD. P < 0.05; p < 0.01. N.s, no significant difference.

FIG. 4 shows DUSP12 overexpression inhibits inflammation and apoptosis in liver IRI injury, NTG and DUSP12-TG mice received liver IRI surgery or sham surgery; wherein:

(A) 6h after IRI, representative HE staining images of ischemic lobes and quantitative analysis of necrotic areas in each group of mice (n ═ 6);

(B) serum ALT and AST levels were determined 6h after ischemia reperfusion or sham surgery in NTG and DUSP12-TG mice (n-8-9);

(C) NTG and DUSP12-TG mice post IRI 6hTNF- α, IL-1 β, IL-6 and CCL 2mRNA levels (n-4);

(D) western blot analysis of activation of NF- κ B signals in NTG and DUSP12-TG mouse livers 6h after ischemia/reperfusion stimulation (n ═ 3);

(E) each group of mice was immuno-fluorescent stained for CD11b positive inflammatory cells (red) (n-5);

(F) representative images and TUNEL staining quantification of liver tissue sections were taken at 6h reperfusion for each group (n-5);

(G) observing the expression levels of liver Bad, Bax and Bcl-2 mrnas 6h after ischemia reperfusion in NTG and DUSP12-TG mice (n-4);

(H) western blot analysis of expression of Bax, C-Caspase3 and Bcl-2 in NTG and DUSP12-TG mice ischemia-reperfusion 6h liver (n ═ 3).

GADPH serves as an internal reference. All data are expressed as mean ± SD. P < 0.05; p < 0.01. N.s, no significant difference.

FIG. 5 shows that deficiency of DUSP12 enhances inflammation and apoptosis in hypoxia/reoxygenation treated hepatocytes, primary hepatocytes from FLOX and DUSP12-CKO mice were stimulated with H/R; wherein:

(A) expression levels of TNF- α, IL-1 β, IL-6 and CCl-2mRNA in hepatocytes after H/R treatment (n ═ 3);

(B) protein levels of the hepatocyte NF- κ B signaling pathway after hypoxic reperfusion (n ═ 3);

(C) mRNA levels of hepatocyte apoptosis-related molecules (Bad, Bax, and Bcl-2) after hypoxic reperfusion (n ═ 3);

(D) protein levels (n-3) of hepatocyte apoptosis-related molecules (Bax and Bcl-2) following hypoxic reperfusion.

All data are expressed as mean ± SD. n is 3 independent experiments. P < 0.01. N.s, no significant difference.

FIG. 6 shows DUSP12 inhibiting ASK1-JNK/p38 signal; wherein:

(A) total and phosphorylated protein levels of ASK1, ERK, JNK and p38 in liver tissue following IRI or Sham stimulation in Flox and CKO mice (n ═ 3);

(B) total and phosphorylated ASK1, ERK, JNK and p38 protein levels (n-3) in liver tissue of mice in ischemia-reperfusion or sham groups;

(C) protein levels of total and phosphorylated ASK1, ERK, JNK, and p38 in hepatocytes and quantification of each index group (n-3).

All data are expressed as mean ± SD. n is 3 independent experiments. P < 0.01. N.s., no significant difference.

Figure 7 is that the effect of DUSP12 deficiency in exacerbating liver IRI injury can be reversed by inhibiting activation of ASK1, wherein:

(A) pretreating DUSP12-CKO and Flox mice by DMSO or GS4997, after 6h of liver IRI, quantitatively analyzing an HE staining image and an ischemic liver lobe necrosis area after 6h of liver ischemia reperfusion, and observing the change of the necrosis area of liver tissues after 6h of liver ischemia reperfusion of the DUSP12-CKO and Flox mice (n is 6);

(B) serum ALT, AST levels (n-8-9) in each group of mice;

(C) levels of TNF- α, IL-1 β, IL-6, CCl-2mRNA (n-4) in each group of mice;

(D) western blot analysis of proteins involved in NF- κ B signal activation (n ═ 3) in each group of liver tissues;

(E) western blot analysis of proteins involved in apoptosis activation (Bax, Bcl2, C-Caspase3) (n-3) in each group of liver tissues;

GADPH was used as an internal reference and all data were expressed as mean ± SD. n is 3 independent experiments. P < 0.05; p < 0.01.

Figure 8 is a graph showing that the effect of DUSP12 on liver IRI is mediated by ASK1 activity, wherein:

(A) expression level of DUSP12, total level of ASK1, JNK and p38 and phosphorylation level (n-3);

(B) relative mRNA levels of hepatocyte cytokines and chemokines (n ═ 3) for each experimental group;

(C) levels of total and phosphorylated IKK β, IkB α and p65 in the indicated group (n-3);

(D) detecting the mRNA expression level (n-3) of each group of apoptosis-related molecules (Bad, Bax, Bcl-2);

(E) the protein expression levels of each of the apoptosis-related molecules Bax and Bcl-2 (n-3) were examined.

All data are expressed as mean ± SD. n is 3 independent experiments. P < 0.05; p < 0.01.

FIG. 9 is a DUSP-12 mouse and DUSP-TG mouse identification, in which:

(A) western blot to detect the levels of DUSP12 protein (n-3) in the livers of Flox and DUSP12-CKO mice;

(B) western blot to detect the levels of DUSP12 protein (n-3) in the livers of NTG and DUSP12-tg mice.

FIG. 10 shows that DUSP12 promotes activation of ASK1/JNK-p38 after I/R injury, 3h and 6h after I/R stimulation, and Western blotting examined total protein and protein levels of phosphorylated ASK1, ERK, JNK and p38 in liver tissues of Flox and CKO mice (n-3).

FIG. 11 is a graph showing that inhibition of ASK1 completely abolishes the damaging effects of DUSP12 deficiency on liver I/R injury, wherein:

(A) immunofluorescence staining (n-3) of CD11b positive inflammatory cells (red) in ischemic liver sections of mice in each group after ASK1 inhibitor was used;

(B) western blot analysis expression of Bax, Bcl2 (n ═ 3) in liver tissues following ASK1 inhibitor;

(C) representative images and TUNEL staining quantification (n-3) were performed on each group of liver tissue sections after ASK1 inhibitor;

(D) western blot assay total protein and protein levels of phosphorylated ASK1, ERK, JNK and p38 (n ═ 3) in liver tissues of Flox and CKO mice after the use of ASK1 inhibitor.

Detailed Description

The following embodiments are incorporated herein. The present invention is further illustrated, and it is understood that these examples are intended only to illustrate the invention and are not intended to limit the scope of the claimed invention, as the following examples illustrate particular experimental conditions and methods, generally in accordance with conventional conditions, not otherwise specified: guidelines for laboratory animal care and use by the national institutes of health; l. speekt et al, scientific press, 2001, cell protocols, etc., or as recommended by the manufacturer.

1. Animal models and procedures

The generation of hepatocyte-specific DUSP12 knockout mice (CKO) and hepatocyte-specific DUSP12 transgenic mice (TG) was similar to previous experiments. CKO born mice (Flox) and TG born mice (NTG) were used as controls. The experiment is carried out in animal experiment center of Wuhan university, referring to the national institutes of health experimental animal care and use guidelines. The study was approved by the ethics committee of the national hospital of Wuhan university. Mice were housed in air-filtered, temperature-controlled (22-24 ℃), humidity-controlled (40-70%) and light-controlled rooms and were allowed free access to a standard diet.

2. Establishment of mouse liver IRI (iron interference) damage model

A mouse liver IRI injury model is constructed by a classical method, and a small amount of water can be drunk by a mouse after fasting for 12 hours before an operation. Mice were anesthetized with 3% sodium pentobarbital prior to surgery, the extremities were fixed in a horizontal position, and the abdominal region hair was removed. The surgical field was disinfected with 10% iodine and 75% ethanol.

A ventral midline incision was made into the abdomen, exposing the hepatic pedicles of the left and middle lobes of the liver. Blocking the portal vein and hepatic artery in the middle, left lobe of the liver with a non-invasive vascular clamp resulted in approximately 70% hepatic ischemia to prevent severe mesenteric venous congestion. After 0.5min, the occluded leaves became white compared to the unoccluded right leaves, indicating successful blood flow occlusion. Then, ischemia was started and maintained for 60 min. Sham group mice were free of liver blood flow blockage, and the vascular clamps were removed after ischemia to restore liver blood flow. After abdominal suturing, the postoperative mice were placed in clean cages and individually placed for observation.

Sampling: mice were removed from Sham surgery group (Sham group) and IRI group at the indicated time post-surgery and anesthetized with 3% sodium pentobarbital. Then, 1mL of blood was taken from the orbital venous plexus, and serum was isolated. Meanwhile, the liver middle lobe is collected and frozen in liquid nitrogen, and the liver left lobe is fixed in a 10% formalin culture medium for 24 hours, dehydrated, embedded and prepared into a paraffin section.

3. Primary hepatocyte separation and construction of hypoxia/reoxygenation (H/R) model

Primary hepatocytes were isolated from liver tissue and control hepatocytes were cultured in complete DMEM/F12 medium containing 10% fetal bovine serum at 37 ℃ and 5% carbon dioxide. For H/R group hepatocytes, 5% CO was used₂And 90% N₂The medium was replaced with equilibrated serum-free DMEM medium and placed in the module incubator of lascona biospherix, n.y., and flushed with the same mixed gas. After 6h, incubate for 6h under normoxic conditions (air/5% CO 2). Cells were collected for further analysis. The specific ASK1 inhibitor GS4997 (S8292; Selleck chemcal, Houston, TX, USA, 80uM) was given to DUSP12-CKO and Flox mice primary hepatocytes 30min prior to hypoxia/reperfusion. In animal models, mice were injected with ASK1 inhibitor or DMSO 30min prior to IRI stimulation.

4. Liver function analysis

Serum alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) concentrations were measured using an ADVIA2400 biochemical analyzer.

5. Pathological analysis

1) HE staining

Liver sections (5 μm) were stained with hematoxylin-eosin (HE) and analyzed for necrotic areas using Image Pro Plus software. And quantitatively analyzed with Image Pro Plus software. The necrotic area of each mouse was quantified as a percentage of the total area of the tissue sections in more than 5 regions.

2) TUNEL staining

For rapid detection of ischemia reperfusion injury induced apoptosis, terminal deoxynucleotidyl transferase (dUTP) nicked end labeling (TUNEL), we used the original apoptosis detection kit (TUNEL)

Plus in situ apoptosis fluorescence detection kit, S7111; ). The TUNEL experiment was performed according to the manufacturer's instructions. Images were obtained under an Olympus DX51 fluorescence microscope. Quantification of TUNEL positive cells per field of tissue sections was performed using Image Pro Plus (version 6.0).

3) Immunohistochemistry

Paraffin section was deparaffinized and hydrated, and C-Caspase-3 immunohistochemical staining was performed. Mouse liver sections were first repaired with bovine serum albumin (BSA or EDTA) for 20min at high temperature. Sections were then incubated with primary antibody (CST9664, USA) overnight at 4 deg.C, rewarmed for 30min at 37 deg.C, washed with PBS, and secondary antibody (Zhongshan Jinqiao, PV9001, China) incubated.

4) Immunofluorescence assay

Immunofluorescent staining was performed with CD11b antibody to detect the degree of infiltration of inflammatory cells in liver sections. After overnight incubation with the CD11b antibody (Boster, BM3925, Wuhan, China, 4 ℃), liver sections were incubated with a secondary antibody (THErmo FisHEr Science, A-11011, Mass.). Nuclei were labeled with DAPI. Immunofluorescence images were collected and analyzed with Image Pro Plus (version 6.0).

5) Immunoblotting (Western blotting)

The protein was collected with protein lysis buffer. Protein samples were treated with loading buffer and separated by 10% SDS-PAGE. Western blot was performed with primary antibody. Adding the secondary antibody of the corresponding strain, and incubating for 1h at room temperature. With the enhanced chemiluminescence system, the results collected were recorded on a photosensitive imaging plate. Images were captured using a gel imaging system (ChemiDoc XRS +). Protein expression levels were determined using Image J software.

6) Real-time quantitative polymerase chain reaction (qRT-PCR)

Extracting total RNA according to the instructions provided by the manufacturer, and using IMPROM-II^TMReverse transcription system (PROM)EGA) synthesis. The relative expression level of each gene was analyzed using beta-actin as an internal reference. Relative mRNA expression levels were calculated and normalized to β -actin.

7) Statistical analysis

Data are expressed as standard deviation of the mean. The SPSS19.0 software was used for the relevant statistical analysis. Significance between the 2 or more groups was determined using one-way anova and Bonferroni analysis (data satisfying homogeneity of variance) or Tamhane's T2 analysis (data demonstrating variance differences). Statistical differences between the two groups were compared using the two-tailed student t-test. The difference was significant when the P value was < 0.05. P values <0.05 and 0.01 are denoted by "+" and "+" respectively.

6. Analysis of results

(1) Liver IRI can induce the expression of DUSP12 to be down-regulated, and the liver IRI damage is aggravated by DUSP12 deficiency

To analyze whether DUSP12 is involved in liver function disorders due to ischemia-reperfusion, we examined the expression of DUSP12 in an in vivo liver IRI injury model and an in vitro H/R model. The results show that expression of DUSP12mRNA and protein was inhibited in an animal model with ischemia 1h reperfusion 6h (fig. 1A, B). The primary hepatocyte protein expression levels continued to decrease 6H after H/R (fig. 1C). Indicating that DUSP12 is involved in the process of liver IRI injury. The significant reduction of expression of DUSP12 in liver IRI injury prompted us to further study the role and mechanism of DUSP12 in liver IRI injury. Therefore, we used hepatocyte-specific DUSP12 knockout mice (DUSP-CKO) to assess their function in liver IRI lesions, and Western blot confirmed successful deletion of DUSP12 in liver CKO mice (fig. 9A). DUSP12-CKO and Flox mice were used to establish a liver IRI injury model. The results showed a significant increase in necrotic area in the CKO group compared to the Flox group (fig. 1D). Serum ALT and AST levels were significantly increased in both groups after 6h of ischemia-reperfusion, and increased in CKO more significantly than in Flox (FIG. 1E). These results indicate that deficiency in DUSP12 exacerbates hepatic IRI injury.

(2) DUSP12 deficiency promotes liver inflammation during liver IRI

Many studies have shown that sterile inflammation is an important marker of IRI of the liver. Therefore, we examined inflammatory responses after IRI in DUSP12-CKO and Flox mice and the corresponding sham groups. Hepatocyte DUSP12 deficiency resulted in upregulation of the mRNA levels of inflammatory cytokines and chemokines, including TNF- α, IL-1 β, IL-6 and CCL-2, following IRI stimulation in mice (fig. 2A). The NF-kB signal channel is a classical inflammation signal channel and plays a key role in the process of liver IRI. In addition, IRI surgery significantly increased phosphorylation of IKK β and p65 during hepatic IRI, down-regulating expression of I κ B α (fig. 2C).

(3) Exacerbation of apoptosis following IRI in vivo liver due to DUSP12 deficiency

Excessive inflammation leads to hepatocyte apoptosis, exacerbating liver dysfunction. Hepatocyte apoptosis and proliferation/regeneration often affect the function and prognosis of the liver following ischemia reperfusion. Immunofluorescence showed that hepatocytes of DUSP12-CKO mice stained more TUNEL-positive than hepatocytes of Flox mice (fig. 3A), hepatocytes of DUSP12-CKO mice stained significantly more TUNEL-positive than hepatocytes of Flox mice (fig. 3A), and hepatocytes of DUSP12 knockout mice stained significantly more apoptotic than hepatocytes of Flox mice (fig. 3A). The pro-apoptotic molecules Bad and Bax mRNA levels were elevated and the anti-apoptotic molecule Bcl-2mRNA levels were reduced (FIG. 3B). Western blot also showed up-regulation of expression of the pro-apoptotic molecule Bax and inhibition of expression of the anti-apoptotic molecule Bcl-2 (FIG. 3C). In addition, immunohistochemical analysis also showed that C-Caspase3 was up-regulated in DUSP12-CKO mouse 12 (FIG. 3D). These results indicate that DUSP12 deficiency promotes apoptosis during liver IRI injury.

(4) DUSP12 overexpression inhibits inflammation and apoptosis in vivo following liver IRI

Hepatocyte-specific DUSP12 transgenic mice (DUSP12-TG) (fig. 9B) were used to verify the function of DUSP12 in a liver I/R injury model. The results show that overexpression of DUSP12 in hepatocytes reduced necrotic area, and reduced serum ALT and AST levels (fig. 4A, B). Mechanistically, overexpression of DUSP12 inhibited the expression of inflammatory factors (tumor necrosis factor- α, IL-1 β, IL-6, and CCL2) (FIG. 4C). Activation of the NF-. kappa.B signaling pathway was inhibited following I/R injury in DUSP12-TG mice (FIG. 4D). Furthermore, DUSP12-TG mice had reduced CD 11-positive neutrophil infiltration following I/R injury (FIG. 4 e). Apoptosis detection by TUNEL method showed that DUSP12 overexpression decreased TUNEL positive cell number and expression level of pro-apoptotic molecules (Bad, Bax, Caspase), and increased mRNA and protein expression of anti-apoptotic molecule Bcl2 (FIGS. 4F-H). These observations indicate that DUSP12 has a protective effect on the liver during liver I/R injury. Has protective effect on IRI injury of liver.

(5) DUSP12 deficiency exacerbates primary hepatocyte inflammation and apoptosis

To further validate whether DUSP12 affected inflammation and apoptosis of hepatocytes, we examined inflammation and apoptosis of H/R-stimulated DUSP12-CKO and Flox mouse primary hepatocytes. The results indicate that DUSP12 lacks the upregulation of primary hepatocyte inflammatory factors (TNF-. alpha., IL-1. beta., IL-6, and CCl-2) that promote H/R stimulation (FIG. 5A). In addition, DUSP12 deficiency enhanced the activation of the hepatocyte NF- κ B pathway under hypoxic/reoxygenation stimulation (FIG. 5B). In terms of apoptosis, DUSP12 lacked upregulation of mRNA and protein expression levels of hypoxia/reperfusion-stimulated primary hepatocyte pro-apoptotic molecules (Bad and Bax) and downregulation of mRNA and protein expression levels of the anti-apoptotic molecule Bcl2 (fig. 5C, D). Taken together, these results further demonstrate that deficiency in DUSP12 exacerbates inflammation and apoptosis in primary hepatocytes after H/R stimulation.

(6) ASK1-JNK/p38 signaling in DUSP12 regulated liver IRI injury

One previous study reported that DUSP12 can modulate liver inflammation in non-alcoholic fatty liver disease (NAFLD) mice through MAPK signaling pathway. MAPK signaling pathways are key pathways that regulate inflammation and apoptosis in a variety of diseases. Therefore, we speculate that DUSP12 might protect liver cells from IRI damage through the MAPK pathway. In our experiments, we found that the lack of DUSP12 upregulated the phosphorylation levels of p38 and JNK, but not ERK, in liver tissue following I/R injury (fig. 6A). We then examined the expression of ASK1 upstream of the MAPK signaling pathway. The results show that a deficiency in DUSP12 increased the expression of p-ASK1 in liver tissue after ischemia reperfusion (fig. 6A). Furthermore, we also examined the involvement of the MAPK signaling pathway in the DUSP12-NTG I/R mouse model. We obtained the opposite result: DUSP12 overexpression inhibited ASK1-JNK/p38 (FIG. 6B). Also, in the primary hepatocyte H/R model, the results showed that DUSP12 lacks the ability to activate ASK1, p-p38 and p-JNK expression, but not p-ERK (fig. 6C). Western blotting examined the time course of this activation in control and hepatocyte-specific DUSP 12-deficient mice. The results show that DUSP12 promotes the activation of ASK1/JNK-p38 after I/R injury, but the activation levels of ASK1, JNK and p38 at 3h and 6h after I/R injury were not significantly different (FIG. 10A). Taken together, these results indicate that DUSP12 reduces hepatic I/R injury by inhibiting the activation of ASK1-JNK/p38 signal, a mechanism that is ASK1 dependent.

(7) ASK1 is a target of DUSP12 for protecting liver IRI injury

To further verify whether ASK1 mediates the role of DUSP12 in liver I/R injury, ASK1 inhibitors were applied in vivo to inhibit activation of ASK 1. The results indicate that ASK1 inhibitors have protective effects on liver damage caused by DUSP12 deficiency, including reduction of the area of liver necrosis, and reduction of serum ALT and AST levels (fig. 7A, B). We also observed that inhibition of ASK1 decreased the secretion of inflammatory factors (fig. 7C), decreased infiltration of CD11B positive neutrophils (fig. 11A), and inhibited the activation of NF- κ B signaling pathway in liver I/R injury caused by DUSP12 deficiency (fig. 7D). In addition, ASK1 inhibitors inhibited hepatocyte apoptosis, including down-regulation of Bax and C-Caspase3 proteins and up-regulation of Bcl2 protein (fig. 7E). In addition, ASK1 inhibitors down-regulated Bad and Bax mRNA expression, up-regulated Bcl2 expression (fig. 11B), and reduced TUNEL-positive cell infiltration in liver I/R injury due to DUSP12 deficiency (fig. 11C). The application of ASK1 inhibitor before liver I/R injury can significantly inhibit the activation of its downstream JNK/p38 signaling pathway caused by DUSP12 deficiency (fig. 11D). The above results indicate that inhibition of ASK1 completely abolishes the damaging effects of DUSP12 on liver I/R injury.

(8) The effect of DUSP12 on liver IRI is mediated by ASK1 activity

We further demonstrated in vivo the role of ASK1 in mediating DUSP12 in the hepatocyte H/R model. Primary hepatocytes were isolated from liver tissue of DUSP12-CKO and Flox mice and confirmed by Western blotting (FIG. 8A). The ASK1 inhibitor GS4997 was used to block the activity of ASK1 and its downstream signaling molecules JNK and p38 (fig. 8A). RT-PCR results showed that DUSP12 deficiency significantly increased the expression of hepatocyte inflammatory factors (TNF-. alpha., IL-1. beta., IL-6 and CCl-2) following H/R stimulation, but this effect could be inhibited by inhibiting the activation of ASK1 (FIG. 8B). Upon H/R stimulation of DUSP 12-deficient hepatocytes, the NF-. kappa.B signaling pathway is activated. However, when ASK1 inhibitors were added prior to H/R stimulation, activation of the NF- κ B signaling pathway was terminated (fig. 8C). In addition, inhibition of ASK1 showed significant protection against hepatocyte apoptosis due to DUSP12 deficiency, as indicated by decreased mRNA expression of Bax and Bad, decreased protein expression of Bax, and increased mRNA and protein expression of Bcl2 (fig. 8D, E). In conclusion, the above results indicate that inhibition of ASK1 can block the effects of DUSP12 deficiency-induced hepatocyte inflammation and apoptosis, etc., suggesting that ASK1 mediates the protective effect of DUSP12 in liver I/R injury.

Claims (2)

1. Application of DUSP12 in preparation of medicines for preventing/treating hepatic ischemia-reperfusion injury.
2. 2. The use of claim 1, wherein the medicament is a medicament capable of promoting expression of DUSP 12.

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