阅读说明：本技术 帕立骨化醇在制备治疗胆汁淤积性肝损伤药物中的新用途 (New application of paricalcitol in preparation of medicine for treating cholestatic liver injury ) 是由 李丽华 翁志勇 于 2020-12-20 设计创作，主要内容包括：本发明公开了一种帕立骨化醇的医药新用途。帕立骨化醇用于治疗胆汁淤积诱导的肝损伤时,能够有效减少肝脏内胆汁的淤积,减轻肝细胞损伤,抑制肝纤维化形成,提高生存率,且治疗有效剂量为50-200ng/kg。(The invention discloses a new medical application of paricalcitol. When the paricalcitol is used for treating hepatic injury induced by cholestasis, the paricalcitol can effectively reduce the cholestasis of the liver, relieve hepatocyte injury, inhibit hepatic fibrosis formation and improve survival rate, and the effective treatment dose is 50-200 ng/kg.)

1. Novel use of paricalcitol in the manufacture of a medicament for the treatment of cholestatic liver injury, wherein said liver injury is induced by cholestasis.

2. The novel use of paricalcitol, as claimed in claim 1, wherein paricalcitol reduces the accumulation of bile in the liver, lowers the serum total bile acid and bilirubin levels; reducing hepatocyte damage, reducing focal necrotic area of liver, and reducing glutamic-pyruvic transaminase level in serum; inhibit the activation of hepatic stellate cells, reduce the formation of hepatic fibrosis caused by collagen deposition; effectively improve survival rate, and the effective dose is 50-200 ng/kg.

3. Use of paricalcitol, according to claims 1-2, for the preparation of a medicament for the treatment of cholestatic liver injury.

Technical Field

The invention relates to treatment of cholestatic liver injury, and in particular relates to a drug paricalcitol.

Technical Field

Human cholestatic liver disease has various etiologies, can cause hepatocyte damage and fibrosis, and can be seriously developed into cirrhosis and liver failure or even death. Clinical investigations have shown that Vitamin D deficiency is closely related to intrahepatic cholestasis during pregnancy [ Song Hong-Bi, Xu Yin, Yang Xiaowu, et al, High prediction of Vitamin D deficiency in pregnant who and its correlation shift with additive prediction out genes in Guizhou, China [ J ]. J. Int Med. Res.2018 Nov;46(11): 4500-. Whereas Vitamin D supplementation may modulate the expression of bile acid regulatory genes and inhibit common bile duct ligation-induced inflammation in mice [ Michitaka Ogura, Shigeru Nishida, Michiyasu Ishizawa, et al Vitamin D3 models of the expression of double acid regulatory genes and expressions in biological product-linked mice [ J ]. J Pharmacol Exp Heat, 2009 Feb;328 (564-70) ]. However, the mechanism is unclear.

Paricalcitol is an analog of 1, 25-dihydroxyvitamin D and is the active form of vitamin D2. In 2005, paricalcitol capsules were marketed in the united states for the treatment of secondary hyperparathyroidism and chronic renal failure. With the intensive research, paricalcitol has a therapeutic effect on brain diseases, such as Alzheimer's disease, Parkinson's disease, heart inflammation and the like. However, it is not known whether paricalcitol can reduce cholestatic liver injury.

Disclosure of Invention

The invention aims to provide a new medical application of paricalcitol, which has the effect of relieving cholestasis-induced liver injury.

The Paricalcitol (paricaltol) for the treatment of cholestatic liver injury is a commercially available product, purchased from Sigma-Aldrich, usa. It is administered by dissolving in glycerol at a dose of 200ng/kg, and is administered by intraperitoneal injection once every other day.

The liver injury is cholestatic liver injury induced by common bile duct ligation.

The preparation method of the animal disease model of cholestatic liver injury induced by bile duct ligation comprises the following steps:

(1) three random groups of C57BL/6 mice were selected as sham-operated, model and treatment groups. When the treated mice were continuously administered paricalcitol for 3 days, three groups of mice were simultaneously started to model. Firstly, placing a mouse in a small animal anesthesia machine for anesthesia (2% isoflurane), fixing the anesthetized mouse on a constant temperature pad at 37 ℃, then performing aseptic laparotomy under a body type dissecting mirror, separating a common bile duct, and threading the common bile duct of the mouse in a sham operation group without ligation; and the two ends of the biliary duct line of the model group and the treatment group of the mice are ligated and cut off in the middle, and then the abdominal cavity is sutured. And (5) returning the mice to the cage for breeding after the mice are awake.

(2) The mice in the treatment group are injected with paricalcitol in the abdominal cavity, the mice in the sham operation group and the mice in the model group are respectively injected with glycerol with the same volume in the abdominal cavity, and the mice are continuously injected for 5 days or 28 days after the model is made.

Based on the established cholestasis-induced mouse cholestasis liver injury disease model, paricalcitol reduces liver injury and improves mouse survival rate by reducing cholestasis of the liver, hepatocyte injury and hepatic fibrosis formation, and is specifically shown in the following steps:

(1) the paricalcitol can reduce the accumulation of bile in the liver. At 2 days after molding, a significant reduction in serum total bile acid and total bilirubin levels was observed with paricalcitol as checked by biochemical methods.

(2) The paricalcitol can reduce cholestasis-induced hepatocyte injury and focal necrotic area of the liver. 2 days after molding, the paricalcitol is observed to obviously reduce the glutamic pyruvic transaminase level in the serum by a serum enzymology inspection method; at 5 days after molding, liver sections were H & E stained by a pathology examination method, and paricalcitol was observed to reduce the area of focal necrosis of the liver.

(3) The paricalcitol can reduce liver fibrosis formation. 5 days and 28 days after the model building, the analysis by a western blot technology shows that the paricalcitol can inhibit the activation of hepatic stellate cells; in addition, using sirius red special staining, paricalcitol was observed to significantly reduce collagen deposition between liver tissues.

(4) The paricalcitol can reduce the mortality rate of mice. After modeling, the survival state of the mice after 28 days of administration is observed and recorded, and statistical analysis shows that the death rate of the mice is obviously reduced by the paricalcitol.

Drawings

In order to more clearly illustrate the technical solution of the present invention, the drawings used in the examples will be briefly described below.

FIG. 1 is a schematic diagram of the experimental technique of the present invention and the statistical analysis results of mouse survival rate in example 1

FIG. 2 is the mouse serological enzymology and liver morphology assay of example 2

FIG. 3 is the analysis of the degree of liver fibrosis in the mouse in example 3.

Detailed Description

The invention is further illustrated with reference to the following figures and specific examples. These examples are to be construed as merely illustrative and not limitative of the remainder of the disclosure in any way whatsoever. After reading the description of the invention, one skilled in the art can make various changes and modifications to the invention, and such equivalent changes and modifications also fall into the scope of the invention defined by the claims.

According to the invention, a choledocholithiasis model of the mouse is established by ligating common Bile Duct (BDL), Paricalcitol (PAL) can effectively reduce cholestasis of the liver, hepatocyte injury and hepatic fibrosis formation, so that liver injury is reduced, the survival rate of the mouse is improved, and the treatment dose is 50-200 ng/kg.

Example 1, protocol and mouse survival analysis

As shown in FIG. 1, experimental mice (C57 BL/6 male mice, 8-10 weeks old, purchased from Beijing Junke Biotechnology Co., Ltd., Jiangsu) were divided into a sham-operated group (administered with PAL and glycerin PG as a solvent, respectively), a model group (administered with PG) and a treatment group (administered with PAL, 200ng/kg, i.p., once every other day). PAL was predosed for 3 days, and at day 4, three groups of mice were concurrently subjected to common bile duct ligation surgery, and then continued for 5 or 28 days (a). Collecting samples of blood, liver and the like of the mouse for subsequent detection. The state of the mice was observed every day, and body weight, hair, feed amount, water amount, number of deaths, and the cause thereof were recorded. Mouse survival was analyzed using statistical analysis software (GraphPad Prism 5). Results (B) show that ligation of common bile duct resulted in nearly 50% mortality in mice. After PAL treatment, the mortality of the mice was significantly reduced, suggesting that PAL improved survival time of the mice by ameliorating liver damage. The invention also discovers that the PAL dose is within 50-200ng/kg, which can effectively improve the survival rate of mice; however, doses above 200 or below 50ng/kg did not improve mouse survival.

Example 2 mouse serological and liver morphological assays

The level of glutamic-pyruvic transaminase (ALT) in serum is an important index for detecting liver injury; while Total Bile Acid (TBA) and Total Bilirubin (TBIL) are important indicators for the detection of hepatic cholestasis. As shown in fig. 2, when the common bile duct of the mice is ligated for 2 days, the ALT, TBA and TBIL levels in the serum are significantly increased, which indicates that the mice cholestatic liver injury model is successfully induced. The levels of these parameters were significantly reduced after PAL treatment. Indicating that PAL was effective in reducing the extent of cholestasis-induced liver damage (a). Mouse livers were fixed, sectioned and stained with hematoxylin and eosin (H & E), and the histological changes were observed under a microscope. After 5 days of common bile duct ligation, bile duct hyperplasia and punctate hepatocyte necrosis foci appear in the area of the junction and around the portal vein; after PAL treatment, the area of punctate hepatocyte necroses was reduced, indicating that PAL mitigated liver damage (B, arrows in the figure indicate necroses).

Example 3 detection of degree of liver fibrosis in mouse

The degree of liver fibrosis was examined by sirius red staining on liver tissue using immunohistochemical techniques. As shown in fig. 3, common bile duct ligation induced activation of hepatic stellate cells, secretion of collagen and deposition around bile ducts, and increased gradually over time. Sirius red dye can specifically stain collagen red. After PAL treatment, red collagen deposition was significantly reduced (A). a-SMA is a specific protein of muscle fiber; the up-regulation of a-SMA protein expression is also a marker for the activation of hepatic stellate cells. Western blot analysis shows that (B) after PAL treatment, the expression of a-SMA protein is obviously reduced, which indicates that PAL pretreatment can effectively inhibit the activation of astrocytes, reduce collagen deposition and prevent liver fibrosis formation.

In conclusion, when the paricalcitol is used for treating cholestasis-induced liver injury, the paricalcitol can effectively reduce cholestasis in the liver, relieve hepatocyte injury, inhibit hepatic fibrosis formation and improve survival rate, and the effective treatment dose is 50-200 ng/kg.