CN116327943A - Application of KLF15 in alcoholic liver disease - Google Patents
Application of KLF15 in alcoholic liver disease Download PDFInfo
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- CN116327943A CN116327943A CN202310108979.3A CN202310108979A CN116327943A CN 116327943 A CN116327943 A CN 116327943A CN 202310108979 A CN202310108979 A CN 202310108979A CN 116327943 A CN116327943 A CN 116327943A
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Abstract
The invention relates to application of KLF15 in alcoholic liver disease, in particular to application of a pharmaceutical composition for increasing KLF15 expression in liver in preparing a medicament for treating alcoholic liver disease, alcoholic liver injury or improving liver cell activity of a subject and a pharmaceutical composition for treating alcoholic liver disease, alcoholic liver injury or improving liver cell activity of the subject. According to the invention, the expression level of the liver KLF15 is regulated, and experiments prove that the oxidation metabolism of ethanol can be promoted by over-expressing the liver KLF15, and the accumulation of alcohol-induced liver lipid and peroxide can be reduced, so that the effects of effectively resisting fatty liver and hepatitis caused by alcohol can be achieved.
Description
Technical Field
The invention belongs to the technical field of biological medicines, in particular relates to application of KLF15 in alcoholic liver diseases, and more particularly relates to prevention and treatment of liver diseases caused by alcohol by using transcription factor KLF15 in the liver. According to the invention, the expression level of the liver Klf15 is increased to prevent and regulate alcoholic liver diseases, and experiments prove that the oxidation metabolism of ethanol can be promoted by increasing the expression level of the KLF15 in the liver, and the accumulation of lipid and peroxide in the liver induced by the ethanol can be reduced, so that the effects of effectively resisting fatty liver and hepatitis caused by the ethanol can be achieved.
Background
Alcoholic liver disease is a liver disease caused by alcohol abuse and has been attracting attention due to its high incidence. About 24 million drinkers worldwide, 9.6 million severe drinkers [1] . About 300 tens of thousands of people die from drinking every year worldwide, accounting for 5.3% of all deaths. According to the world health organization report, alcohol-related cirrhosis accounts for 47.9% of all cirrhosis deaths. Alcoholic liver disease is a progressive disease which can be classified into alcoholic fatty liver, alcoholic hepatitis, liver fibrosis, cirrhosis and hepatocellular carcinoma according to pathological progression. The preparation is reversible in the early stage of alcoholic liver disease (alcoholic fatty liver disease and alcoholic hepatitis), and can be relieved by timely clinical treatment and alcohol withdrawal; once cirrhosis has progressed, liver damage is in an irreversible stage.
One of the main current therapeutic strategies for alcoholic liver disease is to abstain from the urge of alcoholics to drink. Related clinical studies have shown that topiramate, disulfiram, baclofen, naltrexone and acamprosate drugs reduce alcohol intake in alcoholics, but because of the toxic side effects of some drugs, patients need to take carefully in combination with their physical condition, such as liver problems, patients should avoid naltrexone and disulfiram, and renal failure patients should avoid naltrexone because of its ability to promote renal tubular secretion [2] . Meanwhile, the severity of alcoholic liver disease is often related to the severity of malnutrition [3] . Early nutritional therapeutic intervention can improve therapeutic response and alleviate symptoms. Providing sufficient calories, proteins and nutrients to support hepatocyte regeneration in existing metabolic alterations of liver disease, improving to some extent alcoholic liver disease. Therefore, supplementing nutrition is also a common adjuvant therapy.
For some patients with severe alcoholic liver disease, partial clinic can also assist in adoptingSome corticosteroid treatments. Inflammation is reduced by reducing transcription of pro-inflammatory cytokines such as tumor necrosis factor TNF-alpha and interleukin IL-8. Prednisolone can effectively prolong survival rate of patients with severe alcoholic liver disease [4] . However, some patients have contraindications for steroid therapy or are resistant to steroid hormones, and other therapeutic approaches have to be pursued. Approximately 40% of alcoholic hepatitis patients do not respond to corticosteroid therapy [5] . Meanwhile, in view of bad prognosis caused by sepsis and infectious complications that may occur when using steroids, it is necessary to prevent infection of patients when using steroid therapy. Liver transplantation offers the possibility of prolonged survival for patients with end-stage alcoholic liver disease. From the information on liver transplants accumulated in 1968 to 2015, it was known that in all liver transplant cases, the European Liver Transplantation Registry (ELTR) reported a cirrhosis case rate of 46% -55% and a case rate involving alcoholic cirrhosis (Alcoholic cirrhosis) of 9% -44% [6] . Liver transplantation provides a physiologically functional liver for patients, reverses complications of end-stage liver disease, improves survival and quality of life, but it cannot treat potential alcoholism and alcohol dependence, with the possibility of causing recurrence.
Kruppel Like Factor (KLF) is the largest and most important basic transcription factor in eukaryotes, a subfamily of zinc finger transcription factors, and the KLF gene in mammals contains at least 18 family members, designated KLF1-KLF18, respectively, in the order of the order found. All KLF family members contain 3 conserved Cys2/His2 zinc finger structures at their C-terminus, which regulate the expression of genes rich in GC and CACCC promoters [7] . Different KLF family members have different biological functions in cells and play a role in different disease progression.
KLF15 is widely expressed in various tissues including heart, liver, adipocytes, skeletal muscle, etc., KLF15 has been demonstrated to control various biological processes such as cell differentiation and proliferation, embryogenesis, tissue development, growth, etc. physiological processes [8] . KLF15 has been shown to be involved in glucose metabolism, fatty acid metabolism, amino acid metabolism, etcImportant metabolic systems are closely related [9-12] . KLF15 is an important transcription factor activating gluconeogenesis-related genes, and KLF15 can promote uptake of glucose by cardiomyocytes through glucose transporter by regulating insulin secretion [13] . KLF15 inhibits SREBP-1c (sterol regulatory element binding protein 1 c) transcription by interaction with LXR/RXR/RIP140 complex, KLF15 overexpression specifically ameliorates hypertriglyceridemia [14] . The KLF15 gene is an important regulator of muscle fat metabolism.
KLF15 has been shown to regulate the metabolic system of endogenous substances (e.g. steroid hormones and bile acids) and exogenous substances (e.g. drugs) by direct and indirect pathways [15] . The experimental results obtained by RNA-seq, molecular and biochemical methods show that KLF15 widely regulates the expression of enzyme and membrane channel genes of the biochemical system of EXM (Endobiotic and Xenobiotic Metabolism: endogenous and exogenous substances metabolism). KLF15 binds directly to genes such as CYP2B10 (metabolism and detoxification enzyme of clinical drugs), SULT1A1 (metabolism and detoxification enzyme of clinical drugs), CYP2E1 (cytochrome enzyme, biochemical enzyme that detoxifies drugs), etc., and regulates their transcription levels; or indirectly control the expression of a broader spectrum of enzyme and membrane channel genes by binding to the key regulatory transcription factor PXR, CAR, NRF2 of the EXM biochemical system, thereby regulating its expression [15] . KLF15 controls nutrient acquisition, nutrient flux and nutrient utilization.
According to the current clinical research data, single therapeutic drugs and technical means are unlikely to become effective treatment strategies for alcoholic liver disease.
To date, no report has been made on the defense and treatment of alcoholic liver disease by increasing the expression level of KLF15 in the liver.
Disclosure of Invention
The invention aims at providing a method for preventing and treating liver diseases and liver injuries caused by alcohol based on the current state of the art, and particularly relates to a method for reducing liver injuries caused by alcohol accumulation based on increasing liver KLF15 and application thereof.
Specifically, the invention provides the following technical scheme:
in one aspect, the invention provides the use of a pharmaceutical combination for increasing KLF15 expression in the liver for the manufacture of a medicament for treating alcoholic liver disease, alcoholic liver injury or increasing hepatocyte viability in a subject.
In some embodiments, the pharmaceutical composition that increases KLF15 expression in the liver comprises a virus or plasmid that overexpresses KLF15.
In some embodiments, the plasmid comprises a Cas9 or CasRx-containing plasmid.
In some embodiments, the virus is selected from the group consisting of retrovirus, lentivirus, adenovirus, and adeno-associated virus.
In some embodiments, the virus is an adeno-associated virus or adenovirus.
In another aspect, the invention provides a pharmaceutical composition for treating alcoholic liver disease, alcoholic liver injury, or improving hepatocyte viability in a subject, wherein the pharmaceutical composition comprises an adeno-associated virus and an adenovirus that overexpress KLF15.
In some embodiments, the adeno-associated virus is selected from the group consisting of serotype AAV2, serotype AAV5, serotype AAV7, serotype AAV9, and serotype AAV8 adeno-associated virus.
In some embodiments, the adeno-associated virus is a serotype AAV8 adeno-associated virus.
In some embodiments, the adeno-associated virus is single-stranded or self-complementing.
In some embodiments, a liver-specific promoter is introduced into the virus or plasmid.
In some embodiments, the liver-specific promoter is selected from the group consisting of Alb promoter, TBG promoter, apoEHCR-hAAT and Afp promoter.
In some embodiments, the liver-specific promoter is an Alb promoter.
In some embodiments, a reporter gene is also introduced into the virus or treatment.
In some embodiments, the reporter gene is selected from the group consisting of green fluorescent protein, yellow fluorescent protein, red fluorescent protein, and luciferase.
In some embodiments, the reporter is a green fluorescent protein.
In some embodiments, the pharmaceutical composition is in liquid, solid, or semi-solid form, e.g., in the form of a tablet, coated tablet, effervescent tablet, capsule, powder, granule, sugar-coated tablet, lozenge, pill, drop, suppository, emulsion, ointment, gel, tincture, paste, cream, wet dressing, mouthwash, inhalant, oral spray, nasal spray, or injection.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
In some embodiments, the pharmaceutically acceptable carrier includes, but is not limited to, diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, odor masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, viscosity enhancers, antioxidants, preservatives, stabilizers, surfactants, and buffers.
In some embodiments, the pharmaceutically acceptable carrier is selected from diluents, fillers, binders, disintegrants, lubricants, glidants, granulating agents, coating agents, wetting agents, solvents, co-solvents, suspending agents, emulsifiers, sweeteners, flavoring agents, odor masking agents, colorants, anti-caking agents, humectants, chelating agents, plasticizers, viscosity enhancing agents, antioxidants, preservatives, stabilizers, surfactants, and buffers.
In some embodiments, the treatment of liver injury is manifested by a reduction in glutamic pyruvic transaminase, glutamic oxaloacetic transaminase, free fatty acids, triglycerides, cholesterol levels.
In some embodiments, the liver injury is a liver injury caused by alcohol stasis.
In some embodiments, the liver injury comprises liver fibrosis, alcoholic cirrhosis, hepatitis, fatty liver, alcoholic liver injury, liver resection, or liver transplantation.
In some embodiments, the alcoholic liver disease includes alcoholic fatty liver, alcoholic hepatitis, liver fibrosis, cirrhosis, and hepatocellular carcinoma.
In another aspect, the invention provides a method of treating liver injury or increasing hepatocyte viability, comprising administering to a subject an effective amount of an adeno-associated virus that overexpresses KLF15.
In some embodiments, the liver cells are alcohol stimulated.
In some embodiments, KLF15 is specifically overexpressed in the liver by a liver-specific promoter.
In some embodiments, the adeno-associated virus that overexpresses KLF15 is administered by intrahepatic injection or intravenous injection.
In some embodiments, the subject is a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or human.
In some embodiments, the subject is a mouse.
In some embodiments, the subject is a human.
In some embodiments, the adeno-associated virus that overexpresses KLF15 is administered via the portal vein, peripheral vein, or tail vein.
The invention performs the following in vitro and in vivo tests, and the results show that:
(1) The liver KLF15 overexpression is increased, so that liver injury and lipid accumulation caused by alcohol can be resisted;
(2) Increase KLF15 over-expression in liver primary cells and AML cells, and can effectively resist the reduction of cell survival rate caused by alcohol and reduce lipid accumulation.
Experiments prove that the liver KLF15 expression can effectively resist liver injury caused by alcohol by increasing the liver KLF15 expression, and provides a foundation for further preparing drugs for resisting alcoholic liver diseases.
In the invention, the liver injury caused by alcohol can be effectively prevented and treated by means of adeno-associated virus. Adeno-associated virus is AAV8 virus inserted with Alb promoter, and is specifically expressed in liver. Experimental results show that 13 days after intravenous injection of KLF15 over-expressed adeno-associated virus, liver KLF15mRNA increases significantly, indicating that adeno-associated virus starts to act.
Definition of the definition
Alcoholic liver disease: is a liver disease caused by long-term high-volume drinking. Fatty liver is usually manifested in the early stage, and can progress to alcoholic hepatitis, liver fibrosis and cirrhosis.
Lipid vacuoles: in paraffin sections, lipid droplets are dissolved in a lipid solvent such as alcohol or xylene, and thus are in a cavitation form, and sometimes are not easily distinguished from the denaturation of blisters.
Lipid accumulation: the pathological changes of excessive fat accumulation in liver cells are common pathological changes of the liver.
HE staining: hematoxylin-eosin staining is one of the staining methods commonly used in paraffin section techniques. Hematoxylin dye solution is alkaline, and mainly causes chromatin in nuclei and ribosomes in cytoplasm to be purple blue; eosin is an acid dye that primarily reds the cytoplasmic and extracellular matrix components. Can effectively reflect tissue morphology, and is one of the most basic and most widely used technical methods in histology, pathology teaching and scientific research.
Oil red staining: oil red O is a fat-soluble azo dye, is a very strong fat-soluble reagent and fat stain, and can specifically stain neutral lipids such as triglycerides in cells or tissues, and weaker stains such as phospholipids and steroids. When a cell or tissue section is immersed in the oil red O staining solution, the oil red O dissolves in the fat (e.g., lipid droplets) within the cell or tissue, making it red or orange-red.
Alb promoter: liver-specific expressed albumin promoter (ALB).
AAV8 adeno-associated virus: AAV adeno-associated virus of serotype AAV8 is suitable for use in liver, muscle, eye.
Free fatty acids: free fatty acids are one of the substances into which neutral fat is broken down. The concentration of free fatty acid in serum is related to lipid metabolism, glycometabolism, and endocrine function, and is increased by diseases such as diabetes, severe liver disorder, and hyperthyroidism.
Cholesterol: one of the lipids. Cholesterol has a wide range of physiological effects in the body, but when it is in excess, it causes hypercholesterolemia, which adversely affects the body.
Triglycerides: triglycerides are the main energy reservoir of the human body, and can be decomposed according to the requirement of the human body, most tissues can supply energy by utilizing decomposition products of the triglycerides, and meanwhile, tissues such as liver, fat and the like can also synthesize the triglycerides and store the triglycerides in adipose tissues.
Adeno-associated virus: adeno-associated virus (AAV) is a preferred virus for use in gene therapy methods. Wild-type AAVs are relatively small-sized DNA viruses that integrate into the genome of the cells they infect in a stable and site-specific manner. Importantly, no human disease associated with AAV infection has been found to date. Thus, adeno-associated virus (AAV) is the gene therapy vector of choice due to lack of pathogenicity.
TBG promoter: the human thyroxine binds to the globulin promoter and its cellular phagocytosis is hepatocytes.
Afp promoter: the mouse alpha fetoprotein enhancer II is fused with human beta globulin promoter, and the cell phagocytosis is liver cell.
ApoEHCR-hAAT: a chimeric promoter of the hepatocyte control region of human apolipoprotein E and the human α1 antitrypsin promoter.
Primary liver cells: liver primary cells are the main cells of the liver that perform their functions, and many important functions are performed by the performer, such as the decomposition of toxins, the synthesis of urea, the production of all plasma proteins in plasma except several immunoglobulins, etc.; it is also closely related to the occurrence of various diseases of liver such as liver fibrosis, liver cancer, etc.
Drawings
FIG. 1 shows a map of AAV adeno-associated virus, wherein FIG. 1A is AAV-EGFP (control virus) ID number: a map of VB010000-9394 npt; fig. 1B is AAV-shKLF15 (knock down virus) ID number: a map of VB211011-1118 bxg; FIG. 1C is AAV-KLF15 (over-expressed virus) ID number: VB211010-1148jte.https:// www.vectorbuilder.cn/design/retrieve.htmlThe detailed carrier customization information may be queried based on the ID number.
FIG. 2A shows Western Blotting of AAV virus-specific expression; FIG. 2B shows qRT-PCR validation results of AAV virus-specific expression; FIG. 2C shows the specific expression of AAV viruses in the liver.
Figure 3 shows that increasing liver KLF15 expression is effective against alcohol-induced liver injury (PF denotes control fluid diet, EF denotes alcohol fluid diet). FIG. 3A shows that mice over-expressing liver KLF15 with virus have a more ruddy and healthy liver phenotype (liver blushing indicates poor liver status) after modeling of alcoholic liver disease compared to mice injected with negative control virus and mice with liver KLF15 knockdown virus, indicating that liver transcription factor KLF15 has potential regulatory effects in alcoholic liver disease; FIG. 3B shows that mice over-expressing liver KLF15 with virus have lower serum liver damage markers glutamic pyruvic transaminase, glutamic oxaloacetic transaminase after modeling of alcoholic liver disease than mice injected with negative control virus EGFP and mice knocked down liver KLF 15; fig. 3C and 3D show that HE staining and oil red staining results, respectively, show that liver KLF15 overexpressed mice have fewer numbers of lipid vacuoles and smaller lipid vacuole areas than other experimental groups; figures 3E-3G show that liver KLF15 over-expression was lower in lipid cholesterol (TC), triglycerides (TG), free fatty acids (NEFA) compared to the liver of other experimental groups, indicating that liver KLF15 over-expression was resistant to the formation of alcoholic fatty liver, reducing liver damage caused by alcohol.
FIG. 4 shows a map of ADV adenovirus, wherein FIG. 4A is ADV-EGFP (control virus) ID number: a map of VB 150925-10024; fig. 4B is ADV-shKLF15 (knock-down virus) ID number: a profile of VB900083-0072 fqn; FIG. 4C is an ADV-KLF15 (over-expressed virus) ID number: a map of VB210324-1295 xkn.https://www.vectorbuilder.cn/ design/retrieve.htmlThe detailed carrier customization information may be queried based on the ID number.
FIG. 5 shows the results of qRT-PCR validation of ADV virus specific expression.
FIG. 6A shows that stimulation of AML cells with 800mM alcohol for 24h resulted in some degree of loss of cell viability.
Fig. 6B shows that increasing KLF15 expression levels in AML cells of the liver cell line can resist reduced cell viability from different concentrations of alcohol stimulation.
Figures 6C-6E show that increasing KLF15 expression levels in AML cells of the liver cell line can reduce lipid cholesterol (TC), triglyceride (TG), free fatty acid (NEFA) levels in the cells.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
Example 1 liver KLF15 overexpression is resistant to alcohol-induced liver injury
AAV8 adeno-associated virus pAAV [ Exp ] having liver-specific promoter Alb and KLF15 structural gene inserted therein was injected into tail veins of eight-week-old male C57 wild-type mice (Hangzhou source laboratory animal technologies Co., ltd.) using a disposable syringe]-Alb>mKlf15[NM_023184.4]WPRE (VB 211010-1148 jte) (Yun Zhou Biotech (Guangzhou) Co., ltd.) and control virus pAAV [ Exp ]]-Alb>mKlf15[NM_023184.4]WPRE (VB 010000-9394 npt) (Yun Zhou vector personalized design), liver KLF15 knock-down virus pAAV [ miR30 ]]-Alb>EGFP:{mKlf15[shRNA#1]WPRE (VB 211011-1118 bxg) (Yun Zhou vector personalized design) (VB 211011-1118 bxg), viral titres were all diluted 1X 10 with PBS 12 GC/ml (GC/ml is the unit of virus titer), 200 microliter/mouse injection dose, 5 mice in each group, can specifically increase KLF15 over-expression in liver (liver specific promoter Alb mediated targeting liver) within 10-13 days. The virus targeting efficiency is verified by shooting green fluorescent protein carried by viruses in the frozen liver sections by using a laser confocal microscope, and the fact that other tissue organs have no fluorescent expression except the frozen liver sections have strong green fluorescence is found, so that the virus targeting liver effect is excellent. KLF15 expression levels were verified using qRT-PCR and Western Blotting. The verification steps are as follows: grinding 30mg frozen liver in 500 μl of lysate provided in RNA extraction kit (RN 002plus, hayishi Biotech Co., ltd.) and centrifuging to obtain extractThe clear solution was subjected to RNA extraction from liver tissue using an RNA extraction kit (Shanghai Yi Cunninghai Biotechnology Co., ltd., ES, RN002 plus), and cDNA was obtained using a reverse transcription kit (Bao Ri doctor Material technology (Beijing) Co., ltd., takara, RR 037A) and stored at-20 ℃. Using One-Step TB
PrimeScript TM RT-PCR Kit II (Perfect Real Time) (Takara, RR086B, takara Shuzo Co., ltd.) was used to verify the expression level of Klf15 by qRT-PCR and normalized with 18S. The primer sequences are as follows: klf15 (F: ACAGGCGAGAAGCCCTTT, R: ATCTGAGCGGGAAAACCTC), 18S (F: AAACGGCTACCACATCCAAG, R: TACAGGGCCTCGAAAGAGTC), the reaction sequence of qRT-PCR is the incubation phase: 95 ℃ for 10min; and (3) a circulation stage: 40 cycles, 5 ℃,10s,65 ℃ and 30s; melting curve stage: 95 ℃,15s,60 ℃,1min,95 ℃ and 15s; and (3) heat preservation: 95 ℃ for 10min. qRT-PCR results showed that AAV effectively enhanced or knocked down Klf15mRNA expression in the liver. 50mg of frozen liver tissue was ground in RIPA lysate (high) (Shanghai Biyun biotechnology Co., ltd., P0013B) to which PMSF, protease inhibitor and phosphatase inhibitor were added, and after being sufficiently lysed on ice for 30 minutes, the supernatant was centrifuged to obtain total protein. Protein was quantified using BCA kit (shanghai bi yun biotechnology limited, P0012), then protein was denatured by adding protein loading buffer (shanghai yase biotechnology limited, LT 103) and boiling at 99 ℃ for 15 minutes, electrophoresis was performed using 10% SDS-PAGE gel (shanghai yase biotechnology limited, PG 112), after transfer membrane was incubated overnight at 4 ℃ using 1:1000 KLF15 antibody (Ai Bokang (shanghai) trade limited, AB 167192), after membrane washing with TBST was performed using 1:2000 anti-goat secondary antibody (Jiangsu philic biotechnology research center limited, affinity, S0010) for 1h, and development was performed using high sensitivity ECL chemiluminescent kit (su new jetsuga biotechnology limited, P2300) after washing with TBST. Western Blotting results showed that AAV virus specificity increased or decreased liver KLF15 protein levels. Subsequently using an alcoholic liquid food/an isocaloric control liquid food containing 6% alcohol (limited biotechnology of Nantong terlafeiThe company TP4030D/TP 4030C) performs the NIAAA model of alcoholic liver disease, the model comprises an adaptation period of alcoholic fluid food (increased from 0% to 80% alcoholic fluid food) of 5 days, pure alcoholic fluid food (Nanton Telofei Biotechnology Co., ltd., TP4030D/TP 4030C) is used for 10 days from 6 days, a certain dose of 37% ethanol solution (volume microliter of the ethanol solution is 20 times of the weight g of the mouse) is infused into the stomach on 16 days, the maltodextrin solution (4.5 g of the maltodextrin is dissolved in 10ml of physiological saline, volume microliter of the maltodextrin is 20 times of the weight g of the mouse) is taken, the peripheral blood, liver and other tissues are euthanized for 4 hours after the stomach infusion, blood is centrifugated in a procoagulant tube at 3000rpm for 10 minutes, upper serum is taken and stored in a refrigerator at-20 ℃, and the tissues are quickly frozen in a refrigerator at-80 ℃ or stored in 4% paraformaldehyde for 48 hours under light protection after the fixation. Liver specific injury index glutamic pyruvic transaminase (ALT), glutamic pyruvic transaminase (AST), lipid accumulation index free fatty acid (NEFA), triglyceride (TG), cholesterol (TC) were detected using glutamic pyruvic transaminase (GPT/ALT) activity detection kit (BC 1555), glutamic pyruvic transaminase (GOT, BC 1565), free fatty acid (NEFA) assay kit (soja, all of the biological engineering research company, south-jing), triglyceride (TG) assay kit (a 110-1-1, all of the biological engineering research company, south-jing), total cholesterol (TC/TCH) assay kit (a 111-1-1, all of the biological engineering research company, south-jing) and reference group (EGFP) respectively, and liver NEFA, TG, TC of mice using virus-specific liver KLF15 increase was found to be significantly lower than liver KLF15 low-knockdown (kf 15 group) and reference group (EGFP group). Overexpression group: ALT (160.+ -. 1.527U/L), AST (172.333.+ -. 10.269U/L), NEFA (22.35.+ -. 2.0228. Mu. Mol/g protein), TG (11.317.+ -. 1.169mmol/g protein), TC (5.406.+ -. 0.248mmol/g protein); control groups of ALT (252.333+8.667U/L), AST (300.667 + -13.860U/L), NEFA (42.53 + -1.769 μmol/g protein), TG (20.859 + -0.688 mmol/g protein), TC (6.067 + -0.203 mmol/g protein); knock-down group: ALT (384.+ -. 3.786U/L), AST (395.+ -. 2.887U/L), NEFA (57.314.+ -. 2.472. Mu. Mol/g protein), TG (29.313.+ -. 1.393mmol/g protein), TC (5.973.+ -. 0.279mmol/g protein), data were collected using mean.+ -. SEMAnalytical analysis (n=3). HE staining and oil red staining both show that the liver tissue of mice over-expressed with liver KLF15 after alcohol modeling has fewer lipid vacuoles (30% -50% reduction in number) and smaller lipid droplets (50% -60% reduction in lipid droplet area) and has healthier liver tissue morphology, so that liver KLF15 over-expression can resist liver injury caused by alcohol.
Example 2 over-expression of KLF15 in hepatocytes was able to combat alcohol-induced decrease in cell viability and lipid accumulation
Liver AML12 cells (purchased from cell bank of academy of sciences of china, SCSP-550) were plated in 48-well plates uniformly at 5 ten thousand cells/well, cultured for 24h with DMEM containing 10% FBS, and after cell attachment, purified for 4h with serum-free DMEM medium. Then, the culture solution was stimulated with serum-free DMEM medium containing 200, 400 and 800mM absolute ethanol for 24 hours, and 450. Mu.l of serum-free DMEM and 50. Mu.l of cck8 cell proliferation toxicity detection reagent were added to each well and mixed uniformly, and after incubation at 37℃for 3 hours, OD was detected at 450nm absorbance using an enzyme-labeled instrument.
Liver AML12 cells (purchased from cell bank of China academy of sciences, SCSP-550) were spread uniformly in 48-well plates according to 3.5 ten thousand cells/well, and cultured with DMEM medium (Verwent Biotechnology (Nanj) Inc., 087-150) containing 10% FBS (Verwent Biotechnology (Nanj) Inc., 319-005) for 24 hours to adhere the cells, and then, 1X 10 cells were used, respectively 8 GC/ml titer KLF15 overexpressing ADV adenovirus (Yun Zhou Biotech (Guangzhou) Co., ltd.) pAV [ Exp]-EGFP-EF1A>mKlf15[NM_023184.4](VB 210324-1295 xkn) (personalized customization of Yun Zhou company), KLF15 knock-down of the ADV adenovirus pAV [ shRNA ]]-EGFP-U6>mKlf15[shRNA](VB 900083-0072 fqn) (personalized customization by Yun Zhou company) specifically increased and decreased KLF15 expression in cells, respectively, according to a transfection of 500. Mu.l/well for 36h, and control ADV adenovirus pAV [ Exp ] was used]-CMV>EGFP (VB 150925-10024) (personalized customization by Yun Zhou company) was used as a reference group. Virus-induced overexpression and knockdown levels of Klf15 were verified using qRT-PCR, RNA in cells was extracted using RNA extraction kit (shanghai yi fir biotechnology limited, RN002 plus) after 36h of virus transfection, and cDNA was obtained using reverse transcription kit (Takara, RR 037A) and stored at-20 ℃. Using One-Step TB 
PrimeScript TM RT-PCR Kit II (Perfect Real Time) (Takara, RR 086B) was performed to verify the expression level of Klf15 by qRT-PCR and normalized with 18S. The primer sequences are as follows: klf15 (F: ACAGGCGAGAAGCCCTTT, R: ATCTGAGCGGGAAAACCTC), 18S (F: AAACGGCTACCACATCCAAG, R: TACAGGGCCTCGAAAGAGTC), the reaction sequence of qRT-PCR is the incubation phase: 95 ℃ for 10min; and (3) a circulation stage: 40 cycles, 5 ℃,10s,65 ℃ and 30s; melting curve stage: 95 ℃,15s,60 ℃,1min,95 ℃ and 15s; and (3) heat preservation: 95 ℃ for 10min. The verification result shows that the virus effectively induces the overexpression and the knocking down of Klf15.
The effect of altered levels of Klf15 expression at cellular levels on alcohol-induced loss of cell viability was examined using CCK 8. After virus transfection, the cells were replaced with serum-free DMEM medium and purified for 4h. Serum-free DMEM medium (vison biotechnology (south ky) limited, 319-005) containing 200, 400 and 800mM absolute ethanol was then used to stimulate for 24h, respectively, after which 450 μl of serum-free DMEM and 50 μl of cck8 reagent (eastern chemical technology (Shanghai) limited, ck 04) were added to each well and mixed well, and after incubation at 37 ℃ for 3h, OD values were detected at 450nm absorbance using an enzyme-labeled instrument (fig. 2A). Cell viability of KLF15 overexpression (66.6%) was significantly higher than control (47.7%) and knockdown (43%).
Simultaneously, 1X 0.25% Trypsin-EDTA pancreatin (Nanjin Biotechnology Co., ltd., C100C 1) is used for digestion and collection of AML12 cells transfected with ADV virus knockdown/over expression/control KLF15 treated with 400mM alcohol for 24 hours, 400 mu l absolute ethyl alcohol is added to every 200 mu l of cells, grinding is carried out at 4 ℃ for 10 minutes, supernatant is obtained after centrifugation for 10 minutes, and the supernatant and free fatty acid (NEFA) measuring kit (Nanjin Biotechnology Co., ltd., A042-1-1), triglyceride (TG) measuring kit (Nanjin Biotechnology Co., ltd., A110-1-1) and total cholesterol (TC/TCH) measuring kit (Nanjin Biotechnology Co., ltd., A111-1-1) are respectively added with free fatty acid (NEFA), triglyceride (TG) and cholesterol (TC) measuring kit working fluid according to the kit specification, and then the corresponding OD value and cholesterol content (TC) are calculated by using a microplate reader. Experiments showed that KLF15 overexpressing cells had less NEFA, TG, TC than the other two groups. Overexpression group: NEFA (248.312 + -2.029 μmol/L), TG (0.081+ -0.001 mmol/L), TC (0.103+ -0.004 mmol/L); control group: NEFA (354.832 + -9.347. Mu. Mol/L), TG (0.102+0.009 mmol/L), TC (0.153+ -0.003 mmol/L), knock-down group NEFA (444.733 + -5.538. Mu. Mol/L), TG (0.154+ -0.003mmol/L), TC (0.168+ -0.0009 mmol/L), data were statistically analyzed using mean+ -SEM (n=3), indicating that cells have less lipid accumulation. KLF15 overexpression in hepatocytes is resistant to alcohol-induced decrease in cell viability and lipid accumulation.
Sequence(s)
SEQ ID NO. 1Alb promoter sequence (from mouse): CTAGCTTCCTTAGCATGACGTTCCACTTTTTTCTAAGGTGGAGCTTACTTCTTTGATTTGATCTTTTGTGAAACTTTTGGAAATTACCCATCTTCCTAAGCTTCTGCTTCTCTCAGTTTTCTGCTTGCTCATTCCACTTTTCCAGCTGACCCTGCCCCCTACCAACATTGCTCCACAAGCACAAATTCATCCAGAGAAAATAAATTCTAAGTTTTATAGTTGTTTGGATCGCATAGGTAGCTAAAGAGGTGGCAACCCACACATCCTTAGGCATGAGCTTGATTTTTTTTGATTTAGAACCTTCCCCTCTCTGTTCCTAGACTACACTACACATTCTGCAAGCATAGCACAGAGCAATGTTCTACTTTAATTACTTTCATTTTCTTGTATCCTCACAGCCTAGAAAATAACCTGCGTTACAGCATCCACTCAGTATCCCTTGAGCATGAGGTGACACTACTTAACATAGGGACGAGATGGTACTTTGTGTCTCCTGCTCTGTCAGCAGGGCACTGTACTTGCTGATACCAGGGAATGTTTGTTCTTAAATACCATCATTCCGGACGTGTTTGCCTTGGCCAGTTTTCCATGTACATGCAGAAAGAAGTTTGGACTGATCAATACAGTCCTCTGCCTTTAAAGCAATAGGAAAAGGCCAACTTGTCTACGTTTAGTATGTGGCTGTAGAAAGGGTATAGATATAAAAATTAAAACTAATGAAATGGCAGTCTTACACATTTTTGGCAGCTTATTTAAAGTCTTGGTGTTAAGTACGCTGGAGCTGTCACAGCTACCAATCAGGCATGTCTGGGAATGAGTACACGGGGACCATAAGTTACTGACATTCGTTTCCCATTCCATTTGAATACACACTTTTGTCATGGTATTGCTTGCTGAAATTGTTTTGCAAAAAAAACCCCTTCAAATTCATATATATTATTTTAATAAATGAATTTTAATTTATCTCAATGTTATAAAAAAGTCAATTTTAATAATTAGGTACTTATATACCCAATAATATCTAACAATCATTTTTAAACATTTGTTTATTGAGCTTATTATGGATGAATCTATCTCTATATACTCTATATACTCTAAAAAAGAAGAAAGACCATAGACAATCATCTATTTGATATGTGTAAAGTTTACATGTGAGTAGACATCAGATGCTCCATTTCTCACTGTAATACCATTTATAGTTACTTGCAAAACTAACTGGAATTCTAGGACTTAAATATTTTAAGTTTTAGCTGGGTGACTGGTTGGAAAATTTTAGGTAAGTACTGAAACCAAGAGATTATAAAACAATAAATTCTAAAGTTTTAGAAGTGATCATAATCAAATATTACCCTCTAATGAAAATATTCCAAAGTTGAGCTACAGAAATTTCAACATAAGATAATTTTAGCTGTAACAATGTAATTTGTTGTCTATTTTCTTTTGAGATACAGTTTTTTCTGTCTAGCTTTGGCTGTCCTGGACCTTGCTCTGTAGACCAGGTTGGTCTTGAACTCAGAGATCTGCTTGCCTCTGCCTTGCAAGTGCTAGGATTAAAAGCATGTGCCACCACTGCCTGGCTACAATCTATGTTTTATAAGAGATTATAAAGCTCTGGCTTTGTGACATTAATCTTTCAGATAATAAGTCTTTTGGATTGTGTCTGGAGAACATACAGACTGTGAGCAGATGTTCAGAGGTATATTTGCTTAGGGGTGAATTCAATCTGCAGCAATAATTATGAGCAGAATTACTGACACTTCCATTTTATACATTCTACTTGCTGATCTATGAAACATAGATAAGCATGCAGGCATTCATCATAGTTTTCTTTATCTGGAAAAACATTAAATATGAAAGAAGCACTTTATTAATACAGTTTAGATGTGTTTTGCCATCTTTTAATTTCTTAAGAAATACTAAGCTGATGCAGAGTGAAGAGTGTGTGAAAAGCAGTGGTGCAGCTTGGCTTGAACTCGTTCTCCAGCTTGGGATCGACCTGCAGGCATGCTTCCATGCCAAGGCCCACACTGAAATGCTCAAATGGGAGACAAAGAGATTAAGCTCTTATGTAAAATTTGCTGTTTTACATAACTTTAATGAATGGACAAAGTCTTGTGCATGGGGGTGGGGGTGGGGTTAGAGGGGAACAGCTCCAGATGGCAAACATACGCAAGGGATTTAGTCAAACAACTTTTTGGCAAAGATGGTATGATTTTGTAATGGGGTAGGAACCAATGAAATGCGAGGTAAGTATGGTTAATGATCTACAGTTATTGGTTAAAGAAGTATATTAGAGCGAGTCTTTCTGCACACAGATCACCTTTCCTATCAACCCC
SEQ ID NO. 2KLF15 structural gene sequence (source mouse): ATGGTGGACCACCTGCTTCCAGTGGACGAGACCTTCTCGTCACCGAAATGCTCAGTGGGTTACCTAGGGGACAGGCTGGCCAGCCGGCAGCCATACCACATGTTGCCCTCGCCCATCTCGGAGGATGACAGCGATGTCTCCAGCCCCTGCTCTTGTGCCAGCCCTGACTCGCAAGCCTTCTGTTCCTGCTACAGTGCGGGTCCAGGCCCTGAGGCCCAGGGCAGCATCTTGGATTTCCTCCTGTCCCGGGCCACACTGGGCAGTGGTGGTGGCAGTGGAGGTATTGGAGATAGCAGTGGCCCTGTGACCTGGGGATCATGGAGGAGAGCCTCTGTGCCTGTGAAGGAGGAACATTTCTGCTTCCCTGAATTTCTGTCAGGGGACACTGATGACGTCTCCAGGCCCTTCCAGCCTACCCTGGAGGAGATTGAAGAATTCCTGGAAGAGAACATGGAGGCTGAGGTCAAGGAGGCCCCAGAGAACGGTAGCAGGGACCTGGAGACCTGTAGCCAGCTCTCAGCTGGGTCACACCGGAGCCACCTTCATCCAGAGTCTGCTGGGAGAGAGCGCTGTACCCCACCACCAGGTGGCACGAGTGGGGGTGGTGCCCAAAGTGCAGGTGAGGGGCCAGCACATGATGGCCCCGTGCCGGTGCTACTGCAGATCCAGCCTGTTGCTGTGAAGCAGGAGGCAGGTACAGGGCCAGCCTCCCCAGGGCAGGCCCCAGAGAGCGTCAAGGTCGCCCAGCTTCTAGTCAACATCCAGGGGCAGACCTTTGCACTCCTGCCTCAAGTGGTACCATCCTCCAACTTGAACCTGCCCTCAAAGTTTGTGCGAATTGCGCCTGTGCCCATTGCCGCCAAACCTATTGGCTCAGGATCCCTAGGGCCCGGCCCTGCTGGCCTCCTTGTGGGCCAGAAGTTTCCCAAGAACCCAGCAGCAGAACTTCTCAAAATGCACAAATGCACTTTCCCAGGCTGCAGCAAGATGTACACCAAGAGCAGCCACCTCAAGGCCCACCTGCGTCGGCACACAGGCGAGAAGCCCTTTGCCTGCACCTGGCCAGGCTGCGGCTGGAGGTTTTCCCGCTCAGATGAGTTGTCAAGGCACCGGCGATCTCACTCGGGTGTGAAGCCGTACCAGTGTCCCGTGTGCGAGAAGAAATTCGCGCGGAGTGACCACCTCTCCAAACACATCAAAGTGCATCGCTTCCCACGAAGCAGCCGCGCAGTACGCGCCATCAACTGA
SEQ ID NO 3EF1A promoter
GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACCTGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGCCTGGTCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA
SEQ ID NO. 4U6 promoter
GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC
SEQ ID NO 5CMV promoter
GTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTC
cDNA sequence transcribed from 6mKlf15 siRNA with SEQ ID NO
CATTTCTGCTTCCCTGAATTTCTCGAGAAATTCAGGGAAGCAGAAATG
Reference to the literature
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The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (10)
1. Use of a pharmaceutical composition that increases KLF15 expression in the liver in the manufacture of a medicament for treating alcoholic liver disease, alcoholic liver injury or increasing hepatocyte viability in a subject, wherein the subject is a mammal, e.g. a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or a human, preferably the subject is a human.
2. The use of claim 1, wherein the pharmaceutical composition that increases KLF15 expression in the liver comprises an adeno-associated virus or adenovirus that overexpresses KLF15.
3. A pharmaceutical composition for treating alcoholic liver disease, alcoholic liver injury or increasing hepatocyte viability in a subject, wherein the pharmaceutical composition comprises an adeno-associated virus or adenovirus that overexpresses KLF15, wherein the subject is a mammal, such as a mouse, rat, guinea pig, rabbit, cat, dog, monkey, or a human, preferably the subject is a human.
4. The use of claim 2 or the pharmaceutical composition of claim 3, wherein the adeno-associated virus is selected from the group consisting of serotype AAV2, serotype AAV5, serotype AAV7, serotype AAV9, and serotype AAV8 adeno-associated virus, preferably the adeno-associated virus is a serotype AAV8 adeno-associated virus.
5. The use according to claim 2 or the pharmaceutical composition according to claim 3 or 4, wherein a liver specific promoter is introduced into the adeno-associated virus or adenovirus, optionally the liver specific promoter is selected from the group consisting of Alb promoter, TBG promoter, apoEHCR-hAAT and Afp promoter, preferably the liver specific promoter is Alb promoter.
6. The use according to claim 2 or the pharmaceutical composition according to any one of claims 3-5, wherein a reporter gene is also introduced into the adeno-associated virus or adenovirus, optionally selected from the group consisting of green fluorescent protein, yellow fluorescent protein, red fluorescent protein and luciferase, preferably the reporter gene is green fluorescent protein.
7. The pharmaceutical composition according to any one of claims 3-6, wherein the pharmaceutical composition is in liquid, solid or semi-solid form, e.g. in the form of a tablet, coated tablet, effervescent tablet, capsule, powder, granule, dragee, lozenge, pill, drop, suppository, emulsion, ointment, gel, tincture, paste, cream, wet dressing, mouthwash, inhalant, oral spray, nasal spray or injection, preferably the pharmaceutical composition is an injection or inhalant.
8. The use according to any one of claims 1, 2, 4-6 or the pharmaceutical composition according to any one of claims 3-7, wherein the liver injury comprises liver fibrosis, alcoholic cirrhosis, hepatitis, fatty liver, alcoholic liver injury, liver resection and liver transplantation.
9. The use according to any one of claims 1, 2, 4-6 or the pharmaceutical composition according to any one of claims 3-7, wherein the alcoholic liver disease comprises alcoholic fatty liver, alcoholic hepatitis, liver fibrosis, cirrhosis and hepatocellular carcinoma.
10. The use according to any one of claims 1, 2, 4-6 or the pharmaceutical composition according to any one of claims 3-7, wherein the hepatocytes are alcohol stimulated.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115029347A (en) * | 2022-05-11 | 2022-09-09 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating liver and kidney cell fibrosis, recombinant plasmid and virus inhibition |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115029347A (en) * | 2022-05-11 | 2022-09-09 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating liver and kidney cell fibrosis, recombinant plasmid and virus inhibition |
| CN115029347B (en) * | 2022-05-11 | 2024-02-20 | 珠海中科先进技术研究院有限公司 | Molecular monitoring sequence for recognizing and regulating hepatic and renal cell fibrosis, recombinant plasmid and virus inhibition |
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