Application of PTH1R as target in treating or preventing nonalcoholic fatty liver fibrosis

文档序号：493127 发布日期：2022-01-07 浏览：8次中文

阅读说明：本技术 Pth1r作为靶点在治疗或预防非酒精性脂肪性肝纤维化中的应用 (Application of PTH1R as target in treating or preventing nonalcoholic fatty liver fibrosis ) 是由李小英洪汀陆炎熊雪莲陈颖于 2021-11-01 设计创作，主要内容包括：本发明公开了PTH1R作为靶点在治疗或预防非酒精性脂肪性肝纤维化中的应用。本发明通过动物实验证明PTH1R敲除可以降低NASH小鼠血清中谷丙转氨酶和谷草转氨酶的水平,降低NASH小鼠肝组织炎症基因表达的升高。本发明通过细胞实验证明PTH1R的敲除可以显著下降HSC细胞的增殖力,降低HSC细胞中多种细胞活化相关基因的表达,表明PTH1R参与NASH疾病进展中胶原纤维沉积,抑制PTH1R的表达可以显著地减缓或预防NASH肝纤维化、肝硬化的发生。本发明提供了PTH1R作为靶点在治疗或预防NASH肝纤维化中的新用途,为临床上NASH肝纤维化疾病的防治提供了新的策略。(The invention discloses application of PTH1R as a target point in treating or preventing nonalcoholic fatty liver fibrosis. Animal experiments prove that the PTH1R knockout can reduce the level of glutamic-pyruvic transaminase and glutamic-oxalacetic transaminase in serum of a NASH mouse and reduce the increase of liver tissue inflammatory gene expression of the NASH mouse. Cell experiments prove that the PTH1R knockout can obviously reduce the proliferation of HSC cells and reduce the expression of various cell activation related genes in the HSC cells, which indicates that PTH1R participates in the collagen fiber deposition in the progression of NASH diseases, and the inhibition of the expression of PTH1R can obviously slow down or prevent the occurrence of NASH hepatic fibrosis and liver cirrhosis. The invention provides a new application of PTH1R as a target point in treating or preventing NASH hepatic fibrosis, and provides a new strategy for clinically preventing and treating NASH hepatic fibrosis diseases.)

1. Use of an agent that inhibits expression and/or activity of PTH1R, or an agent that inhibits the PTH1R/PTH signaling pathway, for the preparation of a medicament for treating and/or preventing nonalcoholic fatty liver fibrosis.

2. The use of claim 1, wherein the agent that inhibits the expression and/or activity of PTH1R comprises an interfering RNA specific for PTH1R or an inhibitor of PTH 1R.

3. The use of claim 2, wherein the specific interfering RNA for PTH1R is: the sequence of the sense strand is shown as SEQ ID NO: 1, and the sequence of the antisense strand is shown as SEQ ID NO: 2.

4. The use of claim 2, wherein the inhibitor of PTH1R comprises a PTH1R neutralizing antibody.

5. The use of any one of claims 1 to 4, wherein the medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient which is an agent that inhibits the expression and/or activity of PTH1R or an agent that inhibits the PTH1R/PTH signaling pathway.

Technical Field

The invention relates to an application of PTH1R as a target point in treating or preventing nonalcoholic fatty liver fibrosis, belonging to the technical field of biological medicines.

Background

With the prevalence of obesity and lifestyle changes, the incidence of nonalcoholic fatty liver disease (NAFLD) is increasing year by year, threatening the health of the near quarter of the world's population. Fatty liver injury causes hepatocyte peroxidation stress, endoplasmic reticulum stress, etc., leading to hepatocyte death, and further forming a vicious circle of hepatic fat accumulation-hepatic injury-insulin resistance-lipid metabolism disorder. The liver cell injury stimulates inflammatory reaction, chemotactic immune cell infiltration and induces inflammation formation. It has been reported that various mechanisms such as lipid peroxidation, interaction between inflammatory corpuscles and intestinal microorganisms, progenitor cell amplification, adaptive immunity and the like in the process of NAFLD (NAFLD) progression can finally induce Hepatic Stellate Cells (HSCs) to activate or transdifferentiate into myofibroblasts, express smooth muscle actin (alpha-smooth muscle actin, alpha-SMA) and cytoglobin, secrete a large amount of extracellular matrix, especially collagen fibers (type I and type III), and induce liver fibrosis formation. Therefore, the persistence of fatty liver cell injury and inflammatory response will induce the persistence and progression of hepatic fibrosis, and whether the associated hepatic fibrosis is an important indicator of the advanced prognosis of nonalcoholic steatohepatitis (NASH). Liver fibrosis is currently considered to be a dynamic, early reversible course of disease, and inhibition of HSC activation and collagen secretion in a lipotoxic environment is an effective therapeutic strategy. However, there is no well-established drug treatment regimen, especially for patients who progress to liver fibrosis or even cirrhosis, and the lack of early effective intervention will threaten the patient's life. The research and development of anti-hepatic fibrosis drugs aiming at the high risk group and the screening of treatment targets are clinical problems to be solved urgently at the present stage.

Parathyroid hormone type 1 receptors (PTH 1R) are a class B G protein-coupled receptor and are expressed in HSC cells. It has been found that PTH1R, in combination with its ligand parathyroid hormone (PTH), is involved in the regulation of a variety of important pathophysiological processes. The serum PTH of the obese patient is increased, and has an independent positive correlation with the fat content of the human body and the incidence of NAFLD, so the method is one of the prediction factors of the severe obese patient suffering from NASH. Based on the activation mechanism of PTH1R and its regulation and control effects on various diseases, the development of a medicine targeting PTH1R signaling cascade is an important application direction for hepatic fibrosis treatment. However, the mechanism of action of PTH1R in NASH hepatic fibrosis is not clear at present.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: at present, no effective medicine capable of treating NASH hepatic fibrosis and liver cirrhosis exists.

In order to solve the technical problems, the invention provides an agent for inhibiting expression and/or activity of PTH1R and application of the agent for inhibiting PTH1R/PTH signal pathway in preparing a medicament for treating and/or preventing nonalcoholic fatty liver fibrosis.

Preferably, the agent that inhibits PTH1R expression and/or activity comprises a specific interfering RNA of PTH1R or an inhibitor of PTH 1R.

Preferably, the specific interfering RNA of PTH1R is: the sequence of the sense strand is shown as SEQ ID NO: 1(GCACACAGCAGCCAACAUATT) and the sequence of the antisense strand is shown in SEQ ID NO: 2 (UAUGUUGGCUGCUGUGUGCTT).

Preferably, the inhibitor of PTH1R comprises a PTH1R neutralizing antibody.

Preferably, the medicament comprises a pharmaceutically acceptable carrier and an effective amount of an active ingredient which is an agent that inhibits PTH1R expression and/or activity or an agent that inhibits the PTH1R/PTH signaling pathway.

Animal experiments prove that the PTH1R knockout can reduce the level of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) in the serum of the NASH mice at different disease progression stages. Reduce the increase of the liver tissue inflammatory gene expression of the NASH mice. Tissue staining shows that knocking out PTH1R can significantly reduce the degree of liver fibrosis of the NASH mice, and activating PTH1R signal can significantly aggravate the deposition of liver collagen fibers of the NASH mice. Therefore, PTH1R signal activation has important regulation and control effect on preventing or treating NASH hepatic fibrosis.

The cell experiment proves that the expression level of PTH1R in HSC cells is obviously increased in the hepatic fibrosis process. The knockout of PTH1R can significantly reduce the proliferation of HSC cells and reduce the expression of various cell activation-related genes in the HSC cells, such as the secretion of alpha-SMA, type I collagen fibers and the like. Meanwhile, the PTH1R knockout inhibits expression of tumor growth factor beta (TGF beta) of the HSC cells, thereby obviously inhibiting the self-amplification cascade reaction of the HSC cells; conversely, activation of PTH1R signal can promote HSC cell activation, increase cell proliferation capacity, and promote massive collagen fiber secretion. It is demonstrated that PTH1R participates in collagen fiber deposition in the progression of NASH disease, and inhibition of PTH1R expression can significantly slow down or prevent the occurrence of NASH liver fibrosis and liver cirrhosis.

Compared with the prior art, the invention has the beneficial effects that:

the invention provides a new application of PTH1R as a target in treating or preventing NASH hepatic fibrosis, in particular provides a new strategy for clinically preventing and treating NASH hepatic fibrosis diseases by inhibiting HSC cell activation, proliferation and collagen fiber secretion under lipotoxic conditions.

Drawings

FIG. 1 is a graph of PTH1R expression in liver tissue of NASH patients, wherein A, B is a cluster analysis graph and a violin analysis graph of single cell sequencing data of liver fibrosis tissue samples of human beings respectively, and C is an immunofluorescence co-localization staining analysis of PTH1R and fibrosis marker protein alpha-SMA in liver tissue samples of NASH patients;

FIG. 2 is the expression of PTH1R in the NASH liver fibrosis model, wherein A is the mRNA level of PTH1R in PTH1R-GFAP KO mice and their wild control mice liver Primary Hepatocytes (PH), stellate cells (HSC), endothelial cells (LESC), B is the protein level of PTH1R in PTH1R-GFAP KO mice and their wild control mice liver HSC cells, and C is the expression level of PTH1R in PTH intervention mice and intervention control mice liver HSC cells;

FIG. 3 is a graph showing the results of H & E, sirius red staining and masson staining of liver tissues of PTH1R-GFAP KO mice and wild control mice thereof in the NASH liver fibrosis model;

FIG. 4 is a graph comparing the ALT content (A) and AST content (B) in serum of PTH1R-GFAP KO mice and their wild control mice under the NASH hepatic fibrosis model;

FIG. 5 shows mRNA expression levels of PTH1R-GFAP KO mice and their wild control mice liver tissues α -SMA, Col1a1, IL-1 β, TNF- α and IL-6 in the NASH liver fibrosis model;

FIG. 6 is a graph of the results of PTH1R signal-activated H & E, sirius red staining, masson staining, F4/80 and TUNEL in PTH intervention-induced mice and their wild control mice following induction in a NASH liver fibrosis model;

FIG. 7 shows the mRNA expression levels of liver tissues alpha-SMA, Col1a1, IL-1 beta, TNF-alpha and IL-6 of PTH1R signal-activated mice and wild control mice thereof under the intervention of PTH after the induction of the NASH hepatic fibrosis model.

FIG. 8 is a graph showing the proliferation potency of cells in HSC cells in siRNA-PTH1R knock-out group and its control transfection group;

fig. 9 shows the protein expression level of collagen marker protein α -SMA in the siRNA-PTH1R knockout group and its control transfection group (a), the neutralizing antibody PTH1R group and its control group (B) in HSC cells, the mRNA expression level of collagen marker protein α -SMA in the siRNA-PTH1R knockout group and its control transfection group (C), and the mRNA expression level of collagen marker protein Col1a1 in the siRNA-PTH1R knockout group and its control transfection group (D).

Fig. 10 is a graph of the proliferative capacity of PTH-intervened HSC cells and their control cells.

FIG. 11 is the expression levels of type I collagen, α -SMA and TGF β protein in PTH-intervened HSC cells and their control cells;

in each of the above figures, a is a statistical analysis, and the comparison between the two groups shows significant difference, P < 0.05.

Detailed Description

In order to make the invention more comprehensible, preferred embodiments are described in detail below with reference to the accompanying drawings.

Example 1

The embodiment provides an application of PTH1R as a target point in treating or preventing NASH hepatic fibrosis:

(I) construction of animal origin and mouse model

Animal sources:

SPF grade C57BL/6 mice 8 weeks old were purchased from Nanjing university model animal institute (certification number 201603265). The common feed for mice is purchased from the research center of Yangzhou university and has the components of about 70% of carbohydrate, 10% of fat and 20% of protein. A NASH fiberization model feed (cat # A02082002) was purchased from Harlan Teklad. Animals were raised in the Specific Pathogen Free (SPF) grade experimental animal center at the Shanghai academy of sciences of china, and the animal operation in the experimental process followed the guidance opinions about animals to be tested, made by the scientific and technical division of 2006.

Constructing a mouse model:

the Cre/LoxP technology is used for constructing a mouse model for specifically knocking out PTH1R by liver HSC cells: albumin promoter-mediated Cre recombinase (GFAP-Cre) homozygous transgenic mice were constructed by Jackson laboratory introduction (Stock number:016992), U.S.A., PTH1R (flox/flox) transgenic mice, entrusted with the university of Nanjing model animal research institute. Firstly, by using Cas9/RNA system gene targeting technology, gRNA aiming at genes is constructed, and Cas9 protein is guided to cut DNA double strands at a specific site of a crRNA guide sequence target. And a Donor vector carrying a target site homology arm and a conditional knockout element is prepared and is injected together with a Cas9 system, the conditional knockout element is recombined to a target site through homologus recombination after a DNA chain is cut by the Cas9 system, a fertilized egg used for injection is C57BL/6J background, and the born mouse is subjected to gene identification through PCR and sequencing to obtain an F0 generation positive mouse. Then, PTH1R (flox/flox) transgenic mice were crossed with GFAP-Cre transgenic mice to breed PTH1R (flox/+)/GFAP-Cre + gene mice. PTH1R (flox/+)/GFAP-Cre + transgenic mice and PTH1R (flox/flox) transgenic mice are hybridized, and PTH1R (flox/flox)/GFAP-Cre + mice which specifically knock out PTH1R gene in liver are bred and marked as GFAP-PTH1R KO. PTH1R (flox/flox)/GFAP-Cre-mice were additionally used as a control, labeled WT (wild type). Feeding each group of mice with NASH and a control feed thereof to construct a hepatic fibrosis model, reserving a serum sample after intervention, reserving a part of tissues to be soaked in 4% paraformaldehyde for preparing frozen sections and tissue wax blocks, quickly freezing the rest parts with liquid nitrogen, and transferring the frozen sections to a refrigerator of-80 ℃ for freezing and storing for later use.

(II) clinical sample source and detection method

Clinical samples:

human cirrhosis tissue samples were provided by the cooperation of doctor voyage at drugstore hospital affiliated at Nanjing university, incorporated standard: selecting the patient data of the Nanjing tympanites hospital and receiving liver resection operation or gallbladder operation from 2015 9 to 2021 to visit 3. Samples of patients with or without cirrhosis of the liver are clinically and pathologically diagnosed. Subjects had signed informed consent and approved by the ethical committee during sample collection.

The detection method comprises the following steps:

1. primary hepatic HSC cell extraction

Using the liver two-step perfusion method, mice were first immersed in 75% ethanol for 10 seconds of sterilization, and then placed on trays in a clean bench. The abdomen is opened by a cross-shaped incision with scissors, the rib is cut at the uppermost part of the incision, and the thoracic cavity is opened. The small intestine was pulled to the left of the mouse, revealing the inferior vena cava and the portal vein. The inferior vena cava is tied in the thoracic cavity, the needle at the water outlet pipe end of the constant flow pump is inserted into the inferior vena cava from bottom to top, and the needle head is fixed by another thread. The portal vein was cut open and the liver perfused with pre-warmed perfusate 1 at a rate of about 10 mL/min for about 7 minutes. The liver was perfused with pre-warmed perfusate 2 at a rate of about 10 mL/min for a period of about 10 minutes. Taking down liver tissue, centrifuging to obtain cell suspension, and separating by percoll separation method to obtain purified HSC cells, wherein the cell culture condition is DMEM culture medium + 10% fetal calf serum, 37 deg.C, 5% CO₂。

The perfusate has the following formula:

perfusate 1: 33mM KCl, 0.441mM KH₂PO₄，4.17mM NaHCO₃，137.93mM NaCl，0.338mM Na₂HPO₄4.75mg/mL D-glucose, 0.5mM ethylene glycol bis (2-aminoethylether) tetraacetic acid. Adjusted to pH 7.4 with 1mM sodium hydroxide and sterile filtered through a 0.22 μm filter.

Perfusate 2: 3mM CaCl₂，1mM MgCl₂，5.33mM KCl，0.441mM KH₂PO₄，4.17 mM NaHCO₃，137.93mM NaCl，0.338mM Na₂HPO₄4.75mg/mL D-glucose, 0.72% (w/v) bovine serum albumin, 0.005% collagenase type IV. Adjusted to pH 7.4 with 1mM sodium hydroxide and sterile filtered through a 0.22 μm filter.

2. Measurement of AST and ALT in serum

Assays were formulated according to the following table, according to commercial kit instructions:

the calculation formula for serum ALT is as follows:

ALT(U/L)＝(_▲sample tube A/min-_▲Blank tube/min) x K;

wherein, K is 4180 (standard substance is compared with the theoretical K value).

AST calculation is as above.

2. Hematoxylin-eosin (H & E) staining

Liver tissues are fixed by 4% paraformaldehyde for 24 hours, embedded by paraffin, cut into 4-micron sections, dewaxed, stained by hematoxylin, dehydrated, differentiated and mounted, and then subjected to image acquisition and analysis by a microscope.

3. Dyeing with sirius red

The paraffin embedded section of the liver is added with 100 mul of the prepared sirius red dyeing solution (0.5 percent of the sirius red dyeing solution is mixed with the saturated picric acid solution in a ratio of 1: 9, hydrochloric acid is added to adjust the pH to be less than 4), and the mixture is incubated for 2h at 37 ℃. And carrying out image acquisition and analysis by using a microscope after the steps of dehydration, transparency and mounting.

4. Masson staining

The paraffin embedded section of the liver is as above, after dewaxing, the liver is respectively stained by potassium dichromate, hematoxylin, ponceau, phosphomolybdic acid and aniline blue, and washed by ultrapure water, and after dehydration by absolute ethyl alcohol, the liver is sealed and image acquisition and analysis are carried out by using a microscope.

F4/80 fluorescent staining

The paraffin embedded section of the liver is as above, the microwave antigen repair is carried out on the dewaxed citrate buffer solution, F4/80 primary antibody (CST company in America) is dripped to the sealed section at 4 ℃ overnight, the washed secondary antibody is inoculated, DAPI counterstaining is carried out, gradient alcohol dehydration is carried out, xylene is transparent, and the anti-fluorescence quenching sealing agent is used for sealing the section. The fluorescence microscope can select the excitation wavelength range of 450-565 nm and the emission wavelength of 515-565nm (green fluorescence).

TUNEL staining

The paraffin embedded section of the liver is as above, protease K working solution is dripped after dewaxing, equilibrium solution working solution is dripped after rinsing, 50 mu L of TdT enzyme working solution is dripped, and the mixture is covered with a glass slide to react in a wet box at 37 ℃ in a dark place. DAPI counterstaining, and mounting by an anti-fluorescence quenching mounting agent. The fluorescence microscope can select the excitation wavelength range of 450-565 nm and the emission wavelength of 515-565nm (green fluorescence).

7. Fluorescent staining of alpha-SMA

The liver paraffin embedded section is as above, after dewaxing, citrate buffer solution microwave antigen retrieval is carried out, alpha-SMA primary antibody (American CST company) is dripped for overnight at 4 ℃, after washing, secondary antibody dressing culture, DAPI counterstaining, gradient alcohol dehydration, xylene transparency and fluorescence quenching resistant sealing agent sealing piece are carried out. The fluorescence microscope can select the excitation wavelength range of 450-500nm and the emission wavelength of 515-565nm (green fluorescence).

8. Real-time quantitative PCR

Mouse cells and liver tissues were lysed using TRIzol (Invitrogen, usa, cat # 15596018), reverse-transcribed into cDNA according to the reverse transcription kit of TaKaRa, PCR primers were designed using Primer design software Primer 5, and PCR Primer specificity was tested using NCBI website BLAST function. After the sample addition on ice was completed, the reaction plate was placed in a LightCycler 480 fluorescent quantitative PCR instrument with the following reaction parameters:

Procedure	reaction conditions	Time
			Pre-denaturation	95℃	5min
Amplification of	95℃	15s
			(number of cycles: 40)	60℃	1min
	60℃	34s
				95℃	5s
Dissolution curve	65℃	1min
			Cooling down	40℃	30s

Finally, the relative quantification is calculated: GAPDH is used as an internal reference, and a relative quantitative method, namely 2-^ΔΔCTThe relative content of the target gene is calculated by the method.

The RT-PCR primer sequences are as follows:

9. protein immunoblotting technique (Western blots)

Extracting proteins in liver tissues or cells by using RIPA lysate (containing 1mM phosphatase inhibitor and 1mM protein phosphatase inhibitor), performing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), performing membrane transfer (200mA) by a Bio-Rad instrument, sealing by using a sealing agent, and adding primary antibody (dilution ratio) with corresponding concentration into the mixture, wherein the primary antibody is PTH1R (1: 1000), alpha-SMA (1: 1000), Collagen I (1: 1000), TGF beta 1 (1: 1000) and GAPDH (1: 2000); shaking overnight at 4 ℃. After washing, the secondary antibody is applied. Soaking the strip in luminous liquid, then placing the strip in an automatic exposure machine, reading the strip image and storing the strip image according to the name of a sample; the gray values of the bands of each lane of the Image are analyzed by using Image J software, the relative gray values are calculated by using the target protein/internal reference protein, and the comparison between groups is carried out.

10. Statistical method

The results of each group of data are expressed by adopting a mean +/-standard error method; the difference between groups is compared by using one-factor variance analysis, and the least significant difference method is adopted for pairwise comparison. Data analysis was performed using SPSS 22.0 statistical software, with P < 0.05 (two-sided) considered statistically different.

(III) experiment and results

1. Determination of the predominant responsive cell type of PTH1R in the progression of liver fibrosis

As shown in fig. 1, the cluster analysis graph (fig. 1A) and the violin analysis graph (fig. 1B) of biological information analysis based on the single cell sequencing detection technique showed that the expression level of PTH1R in human liver tissue was significantly increased mainly in HSC cells during the course of fibrosis. Immunofluorescence co-localized staining analysis showed that in liver tissue of NASH patients, PTH1R had a significant co-localized relationship with the fibrotic marker protein α -SMA, as shown in fig. 1C. It is suggested that HSC cells are the main cells of PTH1R in the course of NASH liver fibrosis.

Establishment of PTH1R-GFAP KO mouse and PTH intervention activation PTH1R mouse model

Taking liver tissues of adult mice for Western blotting analysis, and comparing the results with those of wild WT mice, the results are shown in FIGS. 2A-B, and the expression of PTH1R protein in primary HSC cells of KO mice is obviously reduced, but not total cells or liver cells; meanwhile, fig. 2C shows that PTH1R expression level in PTH-intervening mouse primary HSCs was significantly higher than that of control mice. The successful establishment of a PTH1R-GFAP KO mouse and a mouse model of PTH intervention-activated PTH1R is demonstrated.

PTH1R-GFAP KO mice are resistant to NASH-induced liver fibrosis

The results of the diet-induced NASH fibrosis model and histological evaluation are shown in FIG. 3, HE tissue staining shows severe steatosis and inflammatory infiltration of liver tissues of a mouse with the NASH model, obvious disorder of liver funiculus structure and formation of juniper, and sirius red staining and masson staining show obvious deposition of collagen fibers and formation of hepatic fibrosis of liver tissues of the mouse with the NASH model. In contrast, collagen deposition was significantly reduced in the liver tissue of PTH1R-GFAP KO mice, thus showing that PTH1R in HSC is a powerful intervention point in NASH liver fibrosis. Meanwhile, the results are shown in FIG. 4, the liver serum liver enzymes ALT and AST of the NASH liver fibrosis model mouse are obviously increased compared with the WT mouse, and the liver enzyme level of the PTH1R-GFAP KO mouse is obviously reduced. Suggesting that the knockout of PTH1R can significantly reduce the liver enzyme level of mice with NASH liver fibrosis.

4. Effect of PTH1R-GFAP KO on the liver inflammatory response in NASH mice

Inflammatory status is an important inducer of NASH liver fibrosis progression. To further assess the impairment changes in lipotoxicity-associated liver inflammation in NASH disease, the expression levels of the inflammatory markers IL-1 β, TNF- α and IL-6 in liver tissue were examined. As shown in FIG. 5, the PCR results show that the mRNA expression levels of liver alpha-SMA, Col1a1, IL-1 beta, TNF-alpha and IL-6 of the mice of the NASH hepatic fibrosis model are obviously increased compared with the expression levels of the mRNA expression of the liver alpha-SMA, Col1a1, IL-1 beta, TNF-alpha and IL-6 of the WT mice, while the levels of the inflammatory genes of the PTH1R-GFAP KO mice are obviously reduced compared with the levels of the inflammatory genes of the control group. These results further demonstrate that PTH1R knockout can effectively inhibit liver inflammation levels in the course of NASH fibrosis.

Hepatic fibrosis injury due to NASH aggravated by PTH1R signal activation

In animal models, PTH intervenes in NASH mice for 12 weeks, and changes in liver fibrosis in liver tissues following activation of PTH1R were examined. The results are shown in fig. 6, the expression level of PTH1R protein in PTH intervention group is obviously increased, HE tissue staining shows that hepatic cord structure is obviously disordered and the formation of the limiting plate is obvious, and sirius red staining and masson staining both show that the collagen staining area of the liver of the PTH1R activation group mouse is obviously increased compared with that of the control group. F4/80 and TUNEL staining showed a significant increase in liver inflammation and hepatocyte apoptosis in mice in the PTH intervention group. It is suggested that PTH1R signal activation further exacerbates NASH liver fibrosis progression.

PTH1R signal activation increases the level of inflammation in the fibrotic process of NASH

The PCR results showed that the mRNA expression levels of IL-1 β, TNF- α and IL-6 in the liver of the PTH 1R-activated mice were significantly higher than those of the NASH-controlled mice, and the results are shown in FIG. 7. These results further demonstrate that PTH1R activation can promote liver inflammation levels in the course of NASH fibrosis.

PTH1R knockout inhibition of HSC cell proliferation, collagen secretion capacity and activation-inducing factor TGF β expression

HSC cells are the primary effector cells of PTH1R signaling during lipo-toxic fibrosis progression. PTH1R in murine HSC cells was knocked out by si-RNA (sense5'-3' (SEQ ID NO: 1): GCACACAGCAGCCAACAUATT; antisense5'-3' (SEQ ID NO: 2): UAUGUUGGCUGCUGUGUGCTT) transfection, and cell proliferation capacity was examined using CCK8 kit, and as a result, PTH1R knock-out significantly reduced the proliferation capacity of HSC cells as shown in FIG. 8. As shown in FIGS. 9A-C, Western blotting tests found that the expression level of alpha-SMA in HSC cells was significantly reduced in the PTH1R knockout group or the PTH1R neutralizing antibody (Novus, cat No. NBP1-40067) group compared with the respective control groups. As shown in fig. 9D, the PTH1R knockout group exhibited a decreased amount of type I collagen secretion compared to the control group. All suggest that the PTH1R knockout can inhibit HSC cells from secreting a large amount of fibrosis factors.

PTH1R Signal activation promoting HSC cell proliferation, collagen secretion Capacity and activation inducing factor TGF beta expression

In an in vitro culture HSC cell model, PTH intervenes to activate PTH1R signal in HSC cells for 24 hours, and cell proliferation capacity is detected by using a CCK8 kit, and the result is shown in FIG. 10, and PTH1R signal activation remarkably promotes the proliferation capacity of human HSC cells. Further, Western blotting detection showed that the expression levels of type I collagen, α -SMA and TGF β protein in the PTH 1R-activated HSC cells were found, as shown in FIG. 11. The above results indicate that abnormal activation of PTH1R can promote TGF β expression, amplify the HSC activation process and promote HSC cells to secrete large numbers of collagen fibers.

The above-described embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention in any way and substantially, it should be noted that those skilled in the art may make several modifications and additions without departing from the scope of the present invention, which should also be construed as a protection scope of the present invention.

Sequence listing

<110> Zhongshan Hospital affiliated to Fudan university

Application of <120> PTH1R as target in treating or preventing nonalcoholic fatty liver fibrosis

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA/RNA