CN111139294A - Application of FDPS in the preparation of drugs for the treatment of nonalcoholic steatohepatitis - Google Patents
Application of FDPS in the preparation of drugs for the treatment of nonalcoholic steatohepatitis Download PDFInfo
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- CN111139294A CN111139294A CN202010008976.9A CN202010008976A CN111139294A CN 111139294 A CN111139294 A CN 111139294A CN 202010008976 A CN202010008976 A CN 202010008976A CN 111139294 A CN111139294 A CN 111139294A
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Abstract
本发明利用高脂高糖饮食诱导的非酒精性脂肪性肝炎（nonalcoholic steatohepatitis，简称NASH）小鼠，揭示了FDPS可作为NASH的潜在治疗靶点，并证实了其拮抗剂阿伦膦酸钠对NASH的治疗作用，为开发NASH的治疗药物提供了新思路。
The present invention utilizes high-fat and high-sugar diet-induced nonalcoholic steatohepatitis (NASH) mice to reveal that FDPS can be used as a potential therapeutic target for NASH, and confirms that its antagonist, alendronate sodium, is effective in treating NASH. The therapeutic effect of NASH provides a new idea for the development of therapeutic drugs for NASH.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to an application of FDPS in preparation of a medicine for preventing and/or treating non-alcoholic steatohepatitis.
Background
Nonalcoholic fatty liver disease (NAFLD) has become one of the most serious public health problems at present, and the global incidence rate is about 25%. With the development of economy in China, the change of dietary habits enables the incidence rate of non-alcoholic fatty liver diseases to rise year by year, and the non-alcoholic fatty liver diseases are in the development trend of low age, and become one of the most important chronic liver diseases in China.
Non-alcoholic steatohepatitis (NASH for short) is a core link of NAFLD, develops on the basis of non-alcoholic liver steatosis (NAFL), is manifested by liver steatosis and lobular inflammation, and further can develop into hepatic fibrosis, liver cirrhosis and even liver cancer. In addition, it is also involved in influencing the development of other chronic diseases such as diabetes, atherosclerosis and the like.
At present, the clinical treatment means for treating NASH is very limited, lifestyle intervention is the basic means for treating NAFLD including NASH at present, but simple life intervention is not enough, and in the aspect of drug treatment, vitamin E, pioglitazone and the like are generally adopted, but the drug effect is very limited, so that a novel efficient drug for effectively treating NASH is urgently needed in clinic.
Farnesyl pyrophosphate synthase (FDPS) is an important branching enzyme in the mevalonate pathway, an intermediate step in the synthesis of cholesterol, which catalyzes the sequential condensation of 5-carbon unit isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate to produce 10-carbon unit yakyl pyrophosphate (GPP) and 15-carbon unit farnesyl pyrophosphate (FPP). The mevalonate pathway is a biochemical pathway necessary for a variety of basic products in animals, including cholesterol, isoprenoids, polyterpenes, and ubiquinones, among others. At present, studies have shown that inhibition of FDPS has an effect of ameliorating cardiovascular system diseases. Clinically, FDPS inhibitors such as bisphosphonates including alendronate are used to treat osteoporosis by mechanisms involving inhibition of the osteoclastic process, maintenance of bone structure, improvement of mineralization, increase of cortical thickness and bone density to increase bone strength, and reduction and prevention of osteoporosis. However, the application of targeting inhibition of FDPS in diseases such as non-alcoholic fatty liver disease and the like has not been studied deeply.
Disclosure of Invention
The technical problem to be solved is as follows: aiming at the urgent need of the current clinic for anti-NASH drugs, the invention provides a novel target point FDPS for treating non-alcoholic steatohepatitis, and provides the application of FDPS as the target point in screening and preparing non-alcoholic steatohepatitis treatment drugs.
In order to achieve the above object, the present invention provides the following technical solutions:
the application of FDPS in screening non-alcoholic steatohepatitis treatment medicines, preparing non-alcoholic steatohepatitis treatment medicines or preparing non-alcoholic steatohepatitis diagnosis medicines.
Further, the drug may be a small molecule compound, an antibody drug, a protein, a nucleic acid molecule, a polypeptide, a lipid, a carbohydrate.
Application of FDPS antagonist in preparing medicine for treating or preventing nonalcoholic steatohepatitis.
Further, the FDPS inhibitor is alendronate sodium, risedronic acid, pamidronic acid, or minodronic acid.
Has the advantages that: the invention discloses that the target inhibition of FDPS is a potential treatment means of NASH for the first time, and proves that the alendronate sodium can effectively relieve NASH and relieve the effects of liver lipid deposition, inflammation and fibrosis, so that the NASH can be used for treating NASH.
Drawings
FIG. 1 shows H & E (left) liver sections of mice fed with maintenance diet (NC) and High Fat Diet (HFD) in example 1 and gene expression level of Fdps in liver in this model (right) measured by qPCR.
FIG. 2 shows hematoxylin-eosin (H & E) staining of liver sections of mice fed with HFD and high fat and high sugar (HFFD) (left) in example 1 and the level of hepatic Fdps gene expression in this model was determined by qPCR.
FIG. 3 shows liver Fdps gene transcription (left) and FDPS protein expression levels (middle and right) of mice overexpressing FDPS by liver using qPCR, Western-blot and immunohistochemical detection techniques in example 1.
FIG. 4 shows the plasma ALT (left) and AST (right) levels of the HFD model mouse with liver overexpressing FDPS in example 1.
FIG. 5 hematoxylin-eosin (H & E) and sirius red staining experiments results of the HFD model of liver over-expression of FDPS in example 1.
FIG. 6 shows the results of qPCR assay of liver inflammation factor in HFD model mice with liver overexpressing FDPS in example 1.
FIG. 7 shows the Western-blot technique used to detect the expression of hepatic stellate cell marker α -SMA in the liver of HFD model mouse with FDPS over-expressed in liver in example 1.
FIG. 8 shows the results of liver knockdown FDPS (AAV-shFdps) and control (AAV-GFP) mice liver Fdps transcription level detection under the HFFD model in example 1.
FIG. 9 shows the plasma ALT levels of liver-knockdown FDPS (AAV-shFdps) and control (AAV-GFP) mice in the HFFD model of example 1.
FIG. 10 results of hematoxylin-eosin staining (H & E) of liver in liver knockdown FDPS (AAV-shFdps) and control (AAV-GFP) mice under HFFD model in example 1.
FIG. 11 shows the hepatic knockdown of the levels of hepatic Triglyceride (TG) and cholesterol (TCH) in FDPS (AAV-shFdps) and control (AAV-GFP) mice in the HFFD model of example 1.
FIG. 12 shows the results of qPCR assay of liver knockdown FDPS (AAV-shFdps) and control (AAV-GFP) mouse liver inflammatory factor in HFFD model of example 1.
FIG. 13 results of liver knockdown FDPS (AAV-shFdps) and control (AAV-GFP) mice liver sirius red staining experiments under HFFD model of example 1.
FIG. 14 variation of body weight, food intake and water intake of mice during administration in example 2.
FIG. 15 shows the results of the measurement of ALT and AST in the plasma of mice after the end of the administration in example 2.
FIG. 16 shows the results of the hematoxylin-eosin staining experiment in example 2.
FIG. 17 is a graph showing the content of Triglyceride (TG) in the liver of the mouse after the end of the administration in example 2.
FIG. 18 shows the qPCR detection results of the inflammatory factor gene and collagen synthesis-related gene in liver in example 2.
Detailed Description
The inventors found that FDPS can be expressed in liver parenchymal cells. The research of the inventor firstly reveals that in NASH mice induced by high-fat high-sugar diet (obvious liver cell damage and liver inflammation exist), the expression level of FDPS of the liver is obviously increased compared with the mice induced by only high fat (no obvious liver cell damage and liver inflammation exist). Over-expression of FDPS in liver of high-fat-fed wild-type mice can remarkably aggravate liver inflammation and fibrosis, and knocking down liver FDPS expression can remarkably reduce liver inflammation and other injuries. Further studies showed that the expression of inflammatory factors increased significantly following overexpression of FDPS in liver parenchymal cells. In NASH mice induced by high-fat high-sugar diet, the over-expression of FDPS in the liver is knocked down, so that the damage of lipid drop, inflammation and the like of the liver can be obviously reduced. Most importantly, the antagonist of FDPS, alendronate sodium, can effectively inhibit liver lipid deposition, inflammatory reaction, stellate cell activation and collagen expression. Thus, targeted inhibition of FDPS is a potential means of treating NASH.
The alendronate sodium is one of the longest application history medicines for treating osteoporosis, and can inhibit the osteoclastic process, maintain the bone structure, improve the mineralization degree, increase the cortical thickness and the bone density so as to improve the bone strength and relieve and prevent the osteoporosis. The embodiment 2 of the invention proves that the alendronate sodium can effectively relieve NASH and alleviate liver lipid deposition, inflammatory reaction and hepatic fibrosis. No study of the therapeutic effect of alendronate sodium on NASH has been published so far.
The following will explain in detail the application of FDPS provided by the present invention in the preparation of a medicament for treating non-alcoholic steatohepatitis. The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.
Example 1
FDPS is a potential target for treating NASH
1. Constructing a NASH model:
model one: male C57BL/6J mice (purchased from Tokyo Wintolite laboratory animal technology, Inc.) at 5 weeks of age were housed in a standard SPF environment (free of special pathogens, thermostatted at 25 deg.C, illuminated for 12 hours intervals). Mice were divided into two groups, one group (n ═ 6) fed on maintenance Diet (NC) and one group (n ═ 6) fed on High Fat (HFD) (high fat Diet purchased from Research Diet, usa, cat # D12492). Freely ingesting drinking water, after 21 weeks, anesthetizing a mouse by chloral hydrate, dissecting the mouse, taking blood plasma, taking one of the livers, quickly freezing by liquid nitrogen, preserving at ultralow temperature, and taking one of the livers to be fixed in formalin.
Model two: male C57BL/6J mice (purchased from beijing weitongli laboratory animals technologies ltd) were housed in a standard SPF environment (no special pathogens, thermostated at 25 ℃, illuminated for 12 hours intervals), one group of mice (n ═ 8) were fed High Fat (HFD) (high fat Diet purchased from Research Diet, usa, cat # D12492), another group of mice (n ═ 9) were fed high fat Diet and fructose-containing water (2.31g fructose/100 mL drinking water) (HFFD), ad libitum access to drinking water, and 21 weeks later, material was drawn according to model 1.
Liver total RNA extraction and qPCR detection: placing a proper amount of liver sample into a homogenizing tube containing ceramic beads, adding 1ml of trozol, and homogenizing for 1 minute; after fully grinding, adding 0.2mL of trichloromethane into each tube, taking the supernatant, adding equal volume of isopropanol, uniformly mixing, centrifuging, removing the supernatant, adding 1mL of 75% ethanol into the precipitate, removing the supernatant, volatilizing the residual solvent at room temperature, and adding DEPC to dissolve RNA. cDNA was prepared by reverse transcription using the Vazyme method and amplified using Fdps primer (Antisense: 5'-GCTGACTGAGAAGGAGCTGG-3'; Sense: 5'-GCCTGGAGCAGTTCTACACA-3').
And (3) test results: as shown in fig. 1, HFD induced massive lipid droplet accumulation in mouse liver compared to NC mice in model one (fig. 1 left), whereas HFFD mouse liver not only had massive lipid droplet accumulation with intraleaflet inflammatory cell infiltration in model two (fig. 2 left), as shown in fig. 2. The transcription level of the Fdps gene in the liver of HFD mice was comparable to that of NC mice (right in FIG. 1), but the transcription level of the Fdps gene in the liver of HFFD-induced mice was significantly higher than that of HFD mice (right in FIG. 2), indicating that the transcription level of Fdps was increased with progression of NASH disease.
2. Overexpression of FDPS in the liver of HFD model mice:
a human FDPS (Gene access number: NM-002004, 1260bp) overexpression plasmid was constructed, and pADM-FH-GFP (Shandong Wei Zhen Biotech Co., Ltd.) was used as a vector, and the vector was encapsulated with adenovirus.
Male C57BL/6J mice (purchased from Tokyo Wintolite laboratory animal technology, Inc.) at 8 weeks of age were housed in a standard SPF environment (free of special pathogens, thermostatted at 25 ℃, illuminated at 12 hour intervals) and induced by HFD. In the molding process, 0.1mL adenovirus wrapped with control plasmid (pADM-FH-GFP) and adenovirus wrapped with human FDPS overexpression plasmid (10) are injected into tail vein simultaneously9PFU/only). After two weeks, materials were taken according to model one and examined.
(1) Detection of alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) enzyme activities (Nanjing institute of bioengineering): according to the kit instruction.
(2) Hematoxylin-eosin staining and NAS scoring criteria: preparing a paraffin section from mouse liver tissues, staining in hematoxylin-eosin dye, and sealing with neutral resin. NAS scoring (NASscore) was performed after scanning under a digital pathology scanner. The NAS scoring standard refers to Chinese non-alcoholic fatty liver disease diagnosis and treatment guidelines, namely (1) hepatic cell steatosis: 0 part (< 5%), 1 part (5% -33%), 2 parts (34% -66%), 3 parts (> 66%); (2) intralobular inflammation (20-fold mirror count necrotic foci): 0min (none), 1min (< 2), 2 min (2-4), 3 min (> 4); (3) ballooning of hepatocytes: score 0 (none), score 1 (rare), score 2 (rare).
(3) Sirius red staining and quantification: the mouse liver tissue is cut into paraffin sections, soaked in sirius red dye for 10 minutes, and sealed by neutral resin. Sections were scanned on a digital pathology section scanner and the Percentage of positive areas (stained areas of collagen fibers) to total liver tissue area (percent of positive areas) was calculated using ImageJ 5.0.
(4) Liver RNA is extracted and is reversely transcribed into cDNA, qPCR amplification is carried out, and relative expression quantity of each gene is calculated by utilizing a delta CT method. The primer sequences are as follows,
murine Tlr2 primer sequence:
Sense 5’-CATCACCGGTCAGAAAACAA-3’(SEQ ID NO.1)
Antisense 5’-ACCAAGATCCAGAAGAGCCA-3’(SEQ ID NO.2)
murine Tlr3 primer sequence:
Sense 5’-ATGATACAGGGATTGCACCC-3’(SEQ ID NO.3)
Antisense 5’-ATAGGGACAAAAGTCCCCCA-3’(SEQ ID NO.4)
mouse Tnf primer sequence:
Sense 5’-ACGGCATGGATCTCAAAGAC-3’(SEQ ID NO.5)
Antisense 5’-AGATAGCAAATCGGCTGACG-3’(SEQ ID NO.6)
murine Cxcl1 primer sequence:
Sense 5’-TGCACCCAAACCGAAGTC-3’(SEQ ID NO.7)
Antisense 5’-GTCAGAAGCCAGCGTTCACC-3’(SEQ ID NO.8)
murine Il6 primer sequence:
Sense 5’-TGATGCACTTGCAGAAAACA-3’(SEQ ID NO.9)
Antisense 5’-ACCAGAGGAAATTTTCAATAGGC-3’(SEQ ID NO.10)
murine Il1b primer sequence:
Sense 5’-GGTCAAAGGTTTGGAAGCAG-3’(SEQ ID NO.11)
Antisense 5’-TGTGAAATGCCACCTTTTGA-3’(SEQ ID NO.12)
murine Ccl2 primer sequence:
Sense 5’-CCTGCTGTTCACAGTTGCC-3’(SEQ ID NO.13)
Antisense 5’-ATTGGGATCATCTTGCTGGT-3’(SEQ ID NO.14)
murine Tmem173 primer sequence:
Antisense 5’-GCAGCATATCTCGGAATCGAA-3’(SEQ ID NO.15)
Sense 5’-CAACATGCTCAGTGGTGCAG-3’(SEQ ID NO.16)
murine Nlrp3 primer sequence:
Sense 5’-ATTACCCGCCCGAGAAAGG-3’(SEQ ID NO.17)
Antisense 5’-CATGAGTGTGGCTAGATCCAAG-3’(SEQ ID NO.18)
mouse Acta2 primer sequence:
Sense 5’-ACTGGGACGACATGGAAAAG-3’(SEQ ID NO.19)
Antisense 5’-GTTCAGTGGTGCCTCTGTCA-3’(SEQ ID NO.20)
murine Col1a1 primer sequence:
Antisense 5’-ACATGTTCAGCTTTGTGGACC-3’(SEQ ID NO.21)
Sense 5’-TAGGCCATTGTGTATGCAGC-3’(SEQ ID NO.22)。
(5) a Western Blot method for detecting the expression of α -SMA in liver includes weighing a small mouse liver specimen, adding the lysate in the weight ratio of tissue block (mg) to the volume of lysate (microliter) 1:10, grinding in tissue grinder, centrifugal separation for 15 min, transferring supernatant to new tube, adding proper amount of Loading Buffer, mixing, heating at 99 deg.C for 10 min to obtain protein sample, preparing 10% acrylamide gel, electrophoresis, wet transfer, sealing in 5% defatted milk powder at room temperature for 1 hr, diluting α -SMA 1:1000 times in solution, incubating at 4 deg.C overnight, diluting in 1:3000 times in diluted secondary antibody, incubating at room temperature for 1.5 hr, washing and exposing.
(6) α -SMA immunohistochemical staining, which is to be performed by slicing mouse liver tissue in paraffin, dewaxing at 60 ℃ for 30 minutes, cooling to room temperature, immersing the slices in xylene for 5 minutes × 3 times, 100% ethanol for 3 minutes × 2 times, 95% ethanol for 3 minutes × 2 times, 70% ethanol for 3 minutes × 1 times, and distilled water for 3 minutes × 1 times in this order, transferring the slices into PBS for 2 minutes, transferring the slices into 200mL of 1 × antigen retrieval solution (pH 6.0), covering the lid of the incubation tank, heating with a microwave oven (set as follows: 800w 3 minutes, 200w 5 minutes, checking the volume of the retrieval solution, ensuring that the tissues are immersed below the liquid level, 800w 10 seconds, 200w 5 minutes), opening the lid, cooling for 15 minutes to room temperature, placing the slices into distilled water for 3 minutes, placing the slices into PBS for 3 minutes, closing the sealing solution for 30 minutes, removing the sealing solution, washing the sealing solution with FDPS antibody (primary antibody) for 4 ℃, washing the slices in PBS for 5 minutes × 3 times the next day, adding the sealing solution diluted by dripping the PBS, incubating at room temperature for 60 minutes, drying, incubating for 2 minutes, and wiping the PBS for 1 minute, and developing the color after overnight.
As a result: qPCR detection shows that the transcription level of liver Fdps is obviously increased in mice injected with FDPS overexpression adenovirus (Ad-Fdps) compared with control mice (Ad-GFP) (on the left side of a figure 3), and the expression level of liver FDPS is obviously increased by Western-blot (in the figure 3) and immunohistochemistry (on the right side of the figure 3), so that the result shows that the human FDPS can be successfully and excessively expressed in the liver by injecting the FDPS overexpression adenovirus.
As shown in figure 4, plasma ALT and AST levels were significantly elevated in FDPS overexpressing mice under HFD induction. ALT and AST levels in blood are important indicators of liver damage, and the results indicate that elevated levels of FDPS expression in the liver can lead to liver damage.
As shown in FIG. 5, the H & E results showed that the inflammatory infiltration in liver lobules was significantly increased, and steatosis and ballooning were not significantly changed in FDPS overexpressing group (Ad-Fdps) mice relative to control virus group (Ad-GFP) mice. The sirius red staining result shows that the staining area of collagen fibers in the FDPS overexpression group is remarkably increased, which indicates that liver fibrosis is aggravated after the liver overexpresses the FDPS.
The qPCR result shows that after the liver specifically over-expresses the FDPS, the transcription level of inflammatory factors (Tlr2, Tlr3, Cxcl1, Ccl2, Tnf, Il1b, Il6 and Nlrp3) is obviously increased, the transcription level of liver fibrosis related genes Acta2 and Col1a1 is also obviously increased (figure 6), and the result shows that the FDPS can activate the transcription of the inflammatory factors and the fibrosis related genes after the liver over-expression, α -SMA is a marker of hepatic fibrosis, and the expression level of the liver α -SMA protein of the mouse over-expressed by the FDPS is obviously increased by Western-blot and immunohistochemical staining (figure 7).
Taken together, FDPS overexpression in the liver of mice aggravates liver inflammatory infiltration and fibrosis, driving the development of NASH.
3. Knockdown of FDPS expression in the liver of HFD and HFFD model mice:
murine FDPS (Gene access number: NM-001253751.1, 1263bp) knock-down plasmids were constructed, and pADM-FH-GFP (Shandong Wei Zhen Biotech, Ltd.) was used as a vector, which was packaged with AAV8, which is an adeno-associated virus.
Male C57BL/6J mice (purchased from Beijing Wittiaxle laboratory animal technology Co., Ltd.) of 5 weeks of age were housed in a standard SPF environment (no special pathogen, constant temperature 25 deg.C, 12 hours interval lighting), high fat diet and fructose-containing water (2.31 g)Fructose/100 mL drinking water) feed (HFFD). After 9 weeks of molding, 0.1ml of adeno-associated virus (n ═ 10) coated with control plasmid (pADM-FH-GFP) and adeno-associated virus (5X 10) coated with murine FDPS knock-down plasmid were injected into caudal vein11vg/m, n is 10). After feeding for 3 weeks, the material was taken and examined.
(1) Detection of alanine Aminotransferase (ALT) activity (Nanjing institute for bioengineering): according to the kit instruction.
(2) Detecting the content of triglyceride in liver: placing a small piece of liver tissue, and weighing (g): volume (mL) ═ 1: 9, adding 9 times of volume of absolute ethyl alcohol, grinding for 1 minute on a tissue grinder, centrifuging, and obtaining a supernatant as a sample to be detected. The detection is carried out according to the standard operation method provided by the Nanjing constructed triglyceride detection kit.
(3) Hematoxylin-eosin staining and NAS scoring.
(4) Sirius red staining.
(5) The liver cDNA was subjected to qPCR amplification, and the relative expression level of each gene was calculated by the Δ CT method.
As a result: qPCR detection shows that liver Fdps transcription level of mice injected with FDPS for knocking down adeno-associated virus (AAV-shFdps) is remarkably reduced compared with control mice (AAV-GFP) (FIG. 8, left), and the result shows that the expression of FDPS can be successfully knocked down in the mouse liver by injecting FDPS for knocking down adeno-associated virus.
Plasma ALT levels in mice were significantly reduced following hepatic FDPS knockdown induced by HFFD (figure 9), suggesting that reduced expression of FDPS in the liver reduces high fat and high sugar induced liver damage.
As shown in fig. 10, H & E results showed that there were fewer liver lipid droplets and fewer inflammatory infiltrates within the leaflets, relative to control virus (AAV-GFP) mice, FDPS knockdown (AAV-shFdps) mice. Furthermore, knocking down FDPS expression could significantly reduce hepatic TG and TCH content (fig. 11).
The qPCR result shows that after the FDPS is knocked down, the transcription level of inflammatory factors (Ccl2, Tnf and Il1b) is reduced, and the transcription level of collagen and fibrosis related Acta2 and Col1a1 genes is also obviously reduced (figure 12), and the result shows that the liver can inhibit the transcription of the inflammatory factors and the collagen synthesis related genes after the FDPS is knocked down.
The results of sirius red staining showed that the staining area of liver collagen fibers was significantly reduced in the mice of the FDPS-knockdown group (fig. 13), indicating that liver fibrosis could be reduced after FDPS knockdown.
In conclusion, the knockdown of the expression of FDPS in the liver of the mouse can reduce liver fat accumulation, relieve liver inflammation and fibrosis, inhibit the development of NASH and play a role in protecting the liver, so that direct evidence is provided for developing FDPS antagonists to treat NASH.
Example 2
Therapeutic effect of sodium alendronate on NASH
Construction of HFFD model: male C57BL/6J mice (purchased from Tokyo Wintolite laboratory animal technology, Inc.) at 5 weeks of age were housed in a standard SPF environment (thermostated at 25 ℃ C., 12 hour intervals of illumination). During molding, the feed was fed with high fat diet and fructose-containing water (2.31g fructose/100 mL drinking water) (HFFD), and the feed was ingested freely.
Alendronate sodium dosing regimen: after 10 weeks of feeding, the group was divided into a solvent Control group (Control/Ctrl) (n ═ 8) and an alendronate sodium administration group (ALN) (purchased from Apexbio, usa under the trade name B6586) (n ═ 9). Mice in the control group and the alendronate administration group were intraperitoneally injected with an equal volume of physiological saline and alendronate solution (1mg/kg, alendronate solid powder dissolved in physiological saline) every one, three, and six days for continuous administration for 7 weeks. HFFD diet was maintained during dosing and mouse body weight, food intake and water intake were recorded weekly. After the administration, the mice were anesthetized and dissected, and the material was taken and tested.
(1) Total RNA was extracted and inflammatory factors were detected by qPCR in the same manner as in example 1.
And detecting the expression of inflammatory factor genes and collagen synthesis related genes in the liver by utilizing qPCR.
Murine Ccl3 primer sequence:
Sense 5’-GTGGAATCTTCCGGCTGTAG-3’(SEQ ID NO.23)
Antisense 5’-ACCATGACACTCTGCAACCA-3’(SEQ ID NO.24)
murine Ccl6 primer sequence:
Sense 5’-CTGGCCTCATACAAGAAATGGA-3’(SEQ ID NO.25)
Antisense 5’-CTGAACTCTCCGATCGCTGG-3’(SEQ ID NO.26)
murine Ccl8 primer sequence:
Sense 5’-GAAGGGGGATCTTCAGCTTT-3’(SEQ ID NO.27)
Antisense 5’-TCTTTGCCTGCTGCTCATAG-3’(SEQ ID NO.28)
murine Ccl24 primer sequence:
Sense 5’-TCCCCATAGATTCTGTGACCA-3’(SEQ ID NO.29)
Antisense 5’-AAACCTCGGTGCTATTGCCA-3’(SEQ ID NO.30)
murine Il18 primer sequence:
Sense 5’-CCTCGAACACAGGCTGTCTT-3’(SEQ ID NO.31)
Antisense 5’-CCTGAAGAAAATGGAGACCTGGA-3’(SEQ ID NO.32)
mouse Adgre1 primer sequence:
Sense 5’-GTCTGTGGTGTCAGTGCAGG-3’(SEQ ID NO.33)
Antisense 5’-GGATGTACAGATGGGGGATG-3’(SEQ ID NO.34)
(2) hematoxylin-eosin staining was performed in the same manner as in example 1.
(3) The activity of glutamate pyruvate transaminase was determined in the same manner as in example 1.
(4) And (4) detecting the content of the triglyceride in the liver according to the instruction provided by the Nanjing established triglyceride detection kit.
As a result: there was no significant difference in body weight, food intake and water intake between the two groups of mice during the dosing period (fig. 14), indicating that the sodium alendronate did not significantly alter the body weight and diet of the mice, indicating no significant toxic side effects.
The kit is used for detecting the levels of the biochemical indicators ALT and AST in the plasma of the mice 7 weeks after administration, and the ALT and AST in the plasma of the mice of the alendronate sodium administration group are obviously reduced compared with those of a solvent control group (figure 15), which shows that the alendronate sodium can protect the liver and has the effect of relieving liver injury.
Meanwhile, after hematoxylin-eosin staining is carried out on mouse liver sections, NAS scores show that the FDPS antagonist alendronate sodium has the effect of obviously relieving liver steatosis and inflammatory infiltration (figure 16).
As shown in fig. 17, the levels of triglycerides in the liver of mice in the sodium alendronate-administered group were significantly reduced compared to the solvent control group, indicating that the FDPS antagonist, sodium alendronate, was able to slow down liver fat deposition.
As shown in fig. 18, the transcription levels of inflammatory factors such as Cxcl2, Il6, Il1b and Il18 were significantly reduced by qPCR detection using FDPS antagonist alendronate sodium, and the expression of collagen gene Col1a1 was also significantly reduced, indicating that alendronate sodium has the effects of relieving liver inflammation and inhibiting collagen deposition.
The results show that the alendronate sodium can reduce steatosis, relieve liver inflammation and inhibit collagen deposition, thereby playing a role in protecting the liver.
From the above examples, it is clear that over-expression of FDPS can increase liver inflammation and liver injury to promote NASH development, while knocking down liver FDPS expression reduces the injury caused by high-fat high-sugar, and the result shows that FDPS is a new target for NASH treatment, and the FDPS antagonist alendronate sodium can effectively protect liver, and slow down steatosis, liver inflammation and collagen deposition.
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<210>11
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>11
<210>12
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>12
<210>13
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>13
cctgctgttc acagttgcc 19
<210>14
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>14
<210>15
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>15
gcagcatatc tcggaatcga a 21
<210>16
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>16
<210>17
<211>19
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>17
attacccgcc cgagaaagg 19
<210>18
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>18
catgagtgtg gctagatcca ag 22
<210>19
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>19
<210>20
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>20
<210>21
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>21
acatgttcag ctttgtggac c 21
<210>22
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>22
<210>23
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>23
<210>24
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>24
<210>25
<211>22
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>25
ctggcctcat acaagaaatg ga 22
<210>26
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>26
ctgaactctc cgatcgctgg 20
<210>27
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>27
<210>28
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>28
<210>29
<211>21
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>29
tccccataga ttctgtgacc a 21
<210>30
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>30
<210>31
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>31
<210>32
<211>23
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>32
cctgaagaaa atggagacct gga 23
<210>33
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>33
<210>34
<211>20
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>34
Claims (4)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010008976.9A CN111139294A (en) | 2020-01-06 | 2020-01-06 | Application of FDPS in the preparation of drugs for the treatment of nonalcoholic steatohepatitis |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010008976.9A CN111139294A (en) | 2020-01-06 | 2020-01-06 | Application of FDPS in the preparation of drugs for the treatment of nonalcoholic steatohepatitis |
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| CN111139294A true CN111139294A (en) | 2020-05-12 |
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| CN202010008976.9A Pending CN111139294A (en) | 2020-01-06 | 2020-01-06 | Application of FDPS in the preparation of drugs for the treatment of nonalcoholic steatohepatitis |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105213407A (en) * | 2015-07-01 | 2016-01-06 | 南京大学 | Zoledronic acid is for the preparation of the purposes of the medicine for the treatment of fatty liver disease |
| WO2017126700A1 (en) * | 2016-01-20 | 2017-07-27 | 芳則 森山 | Inhibitor of activity of vesicular nucleotide transporter |
| CN108135168A (en) * | 2015-05-21 | 2018-06-08 | 凯莫森特里克斯股份有限公司 | CCR2 modulator |
| WO2019148125A1 (en) * | 2018-01-29 | 2019-08-01 | Capulus Therapeutics, Llc | Srebp inhibitors comprising a 6-membered central ring |
-
2020
- 2020-01-06 CN CN202010008976.9A patent/CN111139294A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108135168A (en) * | 2015-05-21 | 2018-06-08 | 凯莫森特里克斯股份有限公司 | CCR2 modulator |
| CN105213407A (en) * | 2015-07-01 | 2016-01-06 | 南京大学 | Zoledronic acid is for the preparation of the purposes of the medicine for the treatment of fatty liver disease |
| WO2017126700A1 (en) * | 2016-01-20 | 2017-07-27 | 芳則 森山 | Inhibitor of activity of vesicular nucleotide transporter |
| WO2019148125A1 (en) * | 2018-01-29 | 2019-08-01 | Capulus Therapeutics, Llc | Srebp inhibitors comprising a 6-membered central ring |
Non-Patent Citations (2)
| Title |
|---|
| 徐广宇等: "双膦酸盐类化合物的研究进展" * |
| 段雪飞等: "慢性肝病与骨质疏松症" * |
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Application publication date: 20200512 |