CN115317485B - Application of isoliensinine and neferine in preparation of anti-hepatic fibrosis drugs - Google Patents
Application of isoliensinine and neferine in preparation of anti-hepatic fibrosis drugs Download PDFInfo
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- CN115317485B CN115317485B CN202211082124.XA CN202211082124A CN115317485B CN 115317485 B CN115317485 B CN 115317485B CN 202211082124 A CN202211082124 A CN 202211082124A CN 115317485 B CN115317485 B CN 115317485B
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- isoliensinine
- neferine
- liver fibrosis
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- hepatic stellate
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- AJPXZTKPPINUKN-FIRIVFDPSA-N Isoliensinine Chemical compound C1=CC(OC)=CC=C1C[C@@H]1C2=CC(OC=3C(=CC=C(C[C@@H]4C5=CC(O)=C(OC)C=C5CCN4C)C=3)O)=C(OC)C=C2CCN1C AJPXZTKPPINUKN-FIRIVFDPSA-N 0.000 title claims abstract description 80
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/47—Quinolines; Isoquinolines
- A61K31/472—Non-condensed isoquinolines, e.g. papaverine
- A61K31/4725—Non-condensed isoquinolines, e.g. papaverine containing further heterocyclic rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Medicines Containing Plant Substances (AREA)
- Medicinal Preparation (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The patent relates to an application of isoliensinine in preparing anti-hepatic fibrosis drugs. The alkaloid components in lotus plumule include plumula Nelumbinis alkali, isoliensinine, plumula Nelumbinis alkali, etc. the animal experiment shows that plumula Nelumbinis alkali can reduce CCl 4 The induced concentration of ALT and AST in the serum of the mouse liver fibrosis model and the concentration of HA, LN, PC-III and IV-C in the serum improve pathological symptoms of liver tissues, have good anti-liver fibrosis effect, and the effect of isoliensinine is superior to that of methylliensinine, while the liensinine HAs no obvious treatment effect on liver fibrosis. Cell experiments show that isoliensinine and methyl liensinine reduce the activity of hepatic stellate cells LX-2 and T6, obviously inhibit the expression of activation indexes of hepatic stellate cells, induce the death of the activated hepatic stellate cells, and have better effect than the methyl liensinine. Therefore, can be used for preparing anti-hepatic fibrosis drugs.
Description
Technical Field
The invention relates to application of isoliensinine and methyl liensinine in preparation of anti-hepatic fibrosis drugs, belonging to the field of traditional Chinese medicine pharmacy.
Background
Liver fibrosis is a repair reaction after chronic liver injury caused by various causes, the course of the disease is prolonged and reversible, and if no active and effective intervention measures are available, the liver fibrosis can progress to liver cirrhosis, further a series of serious complications such as liver failure, hepatic encephalopathy, and gastroesophageal varices rupture and bleeding are caused, great pain and death risk are caused for patients, and great economic burden is brought to society. Liver fibrosis is the outcome of chronic liver disease, the central link of which is the activation of hepatic stellate cells (Hepatic stellate cell, HSC) caused by hepatocyte injury. Activation of hepatic stellate cells can disrupt the balance between extracellular matrix (Extracellular Membrane, ECM) production and degradation of the liver, causing excessive deposition of ECM proteins in the liver, thereby causing liver fibrosis, and even cirrhosis. Modern research has shown that: 20% -40% of patients with liver fibrosis eventually develop cirrhosis and even liver cancer. It is important to investigate the inhibition of activated hepatic stellate cells to find effective anti-hepatic fibrosis drugs, and it has become a hot topic of attention for scholars at home and abroad.
The current clinical treatment of liver fibrosis mainly aims at eliminating etiology including antiviral treatment, alcohol-stopping and weight-losing, eliminating pathogenic factors and liver protection treatment, such as strengthening body resistance and removing blood stasis capsules, turtle shell liver softening tablets, turtle shell decocted pills and the like. They only delay the progression of the disease and do not achieve the goal of curing liver fibrosis. In addition, due to the characteristics of complex compound components, multiple targets and unknown action mechanism, the traditional Chinese medicine composition has long treatment period, different curative effects and easy occurrence of side effects which are difficult to evaluate. The traditional Chinese medicine monomer has the advantages of definite components and development of a specific target therapeutic drug, and is a new direction for developing future liver fibrosis drugs. And the traditional Chinese medicine monomer for treating liver fibrosis clinically only has silybin. However, the silybin has poor oral absorption and low bioavailability, so that the clinical application of the silybin is limited. Therefore, there is a need to find safe and efficient liver fibrosis treatment drugs.
The lotus plumule of Chinese traditional medicine is firstly carried in Shen nong Ben Cao Jing (Shen nong's herbal) and is listed as the top grade. The lotus plumule has the main effects of clearing heart fire, soothing nerves, transporting heart and kidney, astringing essence and stopping bleeding. The liensinine, isoliensinine and neferine are main alkaloid active ingredients. Modern pharmacological studies show that the three have the functions of resisting tumor, protecting cells and organs, resisting oxidation and resisting viruses and treating nervous system diseases. However, little study on liver fibrosis is made, only two cases of effects of neferine on liver are seen, one case of which has a preventive and therapeutic effect on liver toxicity injury caused by APAP, and the other case of which is CCl 4 The induced hepatic fibrosis animal has antioxidant and antiinflammatory effects. Animal and cell experiments show that the plumula Nelumbinis alkali has no liver fibrosis resisting effect, and the liver fibrosis resisting effect of the isoliensinine is superior to that of the plumula Nelumbinis alkali, and the research is reported for the first time.
Disclosure of Invention
The invention aims to deeply analyze a novel compound with anti-liver fibrosis effect in lotus plumule, and provides novel medical application of isoliensinine and methyl liensinine.
The invention relates to an anti-hepatic fibrosis drug which is an important alkaloid effective component extracted from traditional Chinese medicine lotus plumule, and the molecular formula of the anti-hepatic fibrosis drug is C respectively 37 H 42 N 2 O 6 C (C) 38 H 44 N 2 O 6 The structural formula is as follows:
the application of isoliensinine in preparing medicine for treating liver fibrosis is provided.
The application of the isoliensinine in preparing the anti-liver fibrosis medicine is characterized in that the anti-liver fibrosis medicine is a medicine composition composed of an active ingredient isoliensinine and pharmaceutically acceptable auxiliary materials.
The application of the isoliensinine in preparing the anti-hepatic fibrosis medicine is characterized in that the active ingredient of the isoliensinine in the medicine composition accounts for 0.1-99.9% of the total weight.
The application of the isoliensinine in preparing the anti-hepatic fibrosis medicine is characterized in that the dosage form of the pharmaceutical composition is tablets, granules, pills, capsules or injections.
The technical scheme of the invention is that a mouse liver fibrosis model and hepatic stellate cells LX-2 and T6 are adopted, the effects of lotus plumule compounds such as lotus plumule alkali, isoliensinine and methyl lotus plumule alkali on liver fibrosis indexes, hepatic stellate cell activation indexes and the like on the whole and in-vitro cell level of a mouse are compared, new compounds with anti-liver fibrosis effects in the lotus plumule of the traditional Chinese medicine are explored, and important experimental basis is provided for preparing anti-liver fibrosis medicines.
Animal experiments prove that the invention comprises the following steps: isoliensinine and neferine can reduce CCl 4 The induced concentration of ALT and AST and the concentration of HA, LN, PC-III and IV-C in the serum of the mouse liver fibrosis model improve pathological symptoms of liver tissues, have good anti-liver fibrosis effect, and the effect of isoliensinine is superior to that of methylliensinine, while the liensinine HAs no obvious treatment effect on liver fibrosis. The mouse pharmacokinetics analysis method established based on UPLC-MS/MS finds that isoliensinine has better relative bioavailability and high safety compared with methyl liensinine. Cell experiments prove that: the isoliensinine and the methyl liensinine reduce the activity of hepatic stellate cells LX-2 and T6, obviously inhibit the expression of activation indexes of the hepatic stellate cells, induce the death of the activated hepatic stellate cells, and have better effect than the methyl liensinine. The new application of isoliensinine and neferine provided by the invention as the medicines for treating liver fibrosis has great significance for breaking through the bottleneck of liver fibrosis treatment.
Drawings
FIG. 1 is CCl in example 1 of the present invention 4 IHC staining patterns of HE, masson staining and alpha-SMA after molding and administration, wherein 1 is solvent control group, 2 is liver fibrosis model group, 3 is liensinine treatment group, 4 is isoliensinine treatment group, 5 is neferine treatment group, 6 is positive liquid medicineA treatment group of feijin;
FIG. 2 is a bar graph showing the effect of different concentrations of isoliensinine or neferine on LX-2 cell viability in example 3 of the invention;
FIG. 3 is a bar graph showing the effect of different concentrations of isoliensinine or neferine on T6 cell viability in example 3 of the invention;
FIG. 4 is a bar graph showing the effect of different concentrations of isoliensinine or neferine on the mRNA expression levels of the LX-2 cell alpha-SMA, COL1 alpha 1 genes in example 4 of the present invention;
FIG. 5 is a bar graph showing the effect of different concentrations of isoliensinine or neferine on mRNA expression levels of the T6 cell α -SMA, COL1 α1 gene in example 4 of the present invention.
FIG. 6 is a bar graph showing the effect of different concentrations of isoliensinine or neferine on the protein expression levels of LX-2 cell alpha-SMA, COL1 alpha 1 in example 5 of the present invention.
FIG. 7 is a bar graph showing the effect of different concentrations of isoliensinine or neferine on protein expression levels of T6 cell alpha-SMA, COL1 alpha 1 in example 5 of the present invention.
FIG. 8 is a graph of different concentrations of isoliensinine or neferine versus LX-2 cell trypan blue staining in example 6 of the present invention.
Detailed Description
The invention is further described below with reference to the drawings and detailed description. It is to be understood that the following examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1 (in vivo experiment): therapeutic effects of liensinine, isoliensinine, neferine on carbon tetrachloride-induced liver fibrosis model mice, and comparison with the positive drug silibinin.
1.1 animals
Male ICR mice were purchased from Shanghai laboratory animals limited, license number: SCXK (Shanghai) 2022-0025. Animals were fed ad libitum, given standard pellet feed, and after 1 week of adaptive feeding in standard light cycle (12 hours light, 12 hours night), room temperature 22 ℃, constant humidity environment, the experiment was started.
1.2 drugs and Primary Agents
Carbon tetrachloride (CCl) 4 The method comprises the steps of carrying out a first treatment on the surface of the Analytically pure, nanjing chemical reagent Co., ltd.), olive oil (Shandong Luhua group Co., ltd.), plumula Nelumbinis alkali (Shanghai Yes biological Co., ltd.), isoliensinine (benefit of the teaching of China university Guo Qinglong, purity 99.5%), plumula Nelumbinis alkali (Shanghai Yes biological Co., ltd.), silibinin (Shanghai Yes biological Co., ltd.), and CCl 4 Mixing with olive oil at a volume ratio of 1:9 to obtain 10% olive oil, and preparing silybin, plumula Nelumbinis alkali, isoliensinine and methyl plumula Nelumbinis alkali into suspension with 0.4% carboxymethylcellulose sodium.
1.3 major instrumentation
Serum biochemical analyzer, tissue slicer, tissue embedding instrument and DM2000 lycra research grade microscope
1.4 grouping of animals
Animals were randomly divided into six groups: normal group, model group, positive drug silybin group (30 mg/kg), plumula Nelumbinis alkali treatment group (50 mg/kg), isoliensinine treatment group (50 mg/kg), plumula Nelumbinis alkali treatment group (50 mg/kg), 8 of each group.
1.5 animal model building and treatment methods
Five groups of mice other than the normal group were given CCl 4 The olive oil solution was injected intraperitoneally (0.5 ml/kg) 3 times per week for 8 weeks, and silibinin, liensinine, isoliensinine, and neferine were administered 1 time per day for 4 weeks from week 5.
1.6 index detection
After the end of the experiment, each mouse was weighed and anesthetized with 10% chloral hydrate (0.2 ml/20 g) intraperitoneally, and after the orbit was bled, the whole liver was dissected and weighed, and the liver/body weight ratio was calculated. After the blood of the mice is kept stand for 2 hours, the mice are centrifuged for 15 minutes at 3000 rpm, and the supernatant is taken and stored at the temperature of minus 20 ℃ for standby. The content of glutamic pyruvic transaminase (ALT), glutamic oxaloacetic transaminase (AST) and alkaline phosphatase (ALP) in the serum of mice was detected by using a semi-automatic biochemical analyzer, and the content of Hyaluronic Acid (HA), laminin (LN) and type III Procollagen (PCIII) in the serum of mice was detected by using an ELISA kit. Liver 4% paraformaldehyde was fixed, and the effects of liver fibrosis liver pathology and collagen deposition were observed by HE, masson staining of tissues and IHC staining of alpha-SMA.
1.7 statistical methods
Experimental data were analyzed using SPSS software (version 20.0), all data expressed in SD or SEM. Group difference analysis was performed using Prism 8.0 (Graph Pad Software Inc, san Diego, CA). The difference of P <0.05 is statistically significant.
1.8 conclusion of results
Results (1): the isoliensinine, the neferine and the silybin, a positive drug, can reduce the ALT and AST content in serum of mice with liver fibrosis, and the isoliensinine has better effect than the neferine, but the neferine has no obvious effect (Table 1).
Table 1 detection results (mean.+ -. Standard deviation) units (U/L) of serum indices ALT and AST of mice in each group
*p<0.05，**p<0.01，***p<0.001, which is, compared with the control group, # p<0.05， ## p<0.01， ### p<0.001, compared to a liver fibrosis model group.
Results (2): isoliensinine, methyl liensinine and silybin, a positive drug, can reduce the content of fibrosis index HA, LN, PCIII in liver fibrosis mouse serum, the effect of isoliensinine is better than that of methyl liensinine, however, no obvious effect of liensinine is seen (table 2).
Table 2 detection results units of serum liver fibrosis index HA, LN, PC-III, IV-C of mice of each group (mg/mL)
*p<0.05，**p<0.01，***p<0.001, which is, compared with the control group, # p<0.05， ## p<0.01， ### p<0.001, compared to a liver fibrosis model group.
Results (3): HE stainingAnalysis of Masson staining and immunohistochemical alpha-SMA staining showed CCl 4 The induced liver fibrosis model of the mice has the characteristics of liver structural disorder, large amount of collagen deposition, obvious liver fibrosis lesion and large amount of hepatic stellate cell activation. Isoliensinine, neferine and silybin, a positive drug, can inhibit collagen deposition in fibrotic liver and inhibit hepatic stellate cell activation indicators, but liensinine does not have this effect (figure 1).
Conclusion: compared with methyl lotus plumule, the isoliensinine plays a better role in resisting hepatic fibrosis.
Example 2 (in vivo experiment): pharmacokinetic study of isoliensinine and neferine in mice
2.1 animals
Male ICR mice were purchased from Shanghai Laek laboratory animal Co., ltd., license number: SCXK (Shanghai) 2022-0025. Animals were fed ad libitum, given standard pellet feed, and after 1 week of adaptive feeding in standard light cycle (12 hours light, 12 hours night), room temperature 22 ℃, constant humidity environment, the experiment was started.
2.2 grouping of animals
Animals were divided into two groups, isoliensinine, neferine treated groups of 10 animals each.
2.3 pharmaceutical treatment
The mice after 12h of fasting were taken and suspension was prepared from isoliensinine and neferine with 0.4% sodium carboxymethylcellulose, and respectively administered by gavage (50 mg/kg).
2.4 sample collection
The mice in the gavage group were bled from the orbit prior to dosing (0 min), 0.083,0.25,0.50,0.75, 1.00,2.00,3.00,4.00,5.00,6.00,8.00, 12.00, 24.00h post-dosing. Plasma samples were pre-treated with 1:2 protein precipitation, methanol was chosen as protein precipitant. mu.L of mouse plasma sample was aspirated, 50. Mu.L of internal standard stock solution (100 ng/mL) was added to a 2mL centrifuge tube, vortexed for 5min, centrifuged at 14000r/min for 10min and 50. Mu.L of supernatant was taken into a liquid phase vial, and 2. Mu.L was used for UPLC-MS/MS analysis.
2.5 Experimental methods
ChromatographyConditions are as follows: using Waters AcquityBEH C18 column (2.1X105 mm,1.8 μm); gradient elution was performed with 0.1% formic acid in water as mobile phase A and 0.1% acetonitrile in water as mobile phase B (0-1 min:10% B,1-3min:10% -80% B,3.0-3.5min:80% -15% B,3.5-5.0min:15% -10% B), total run time: 5min; flow rate: the column temperature was 40℃at 0.3mL/min, and the sample injection amount was 2. Mu.L. Mass spectrometry conditions: adopting an electrospray ion source (ESI), an anion mode, a scanning mode being a multi-reaction detection (MRM) mode, and the ions used for detection are: m/z 557.4/203.18 (isoliensinine, neferine), m/z 248.2/121 (IS). The MS working parameters are set as follows: the spray voltage was 3500V, the ion source temperature was 350 ℃, the dry gas (N2) flow rate was 10L/min, the atomizer pressure 40psi, the sheath gas temperature was 350 ℃, and the sheath gas flow rate was 11L/min. The fragmentation voltages of AST01 and IS were 50V and 260V, respectively, and the collision energies were 34V and 44V, respectively. Data acquisition and analysis was performed using Agilent MassHunter companion software (Multiquant 2.1).
2.6 data processing and statistical analysis
The concentration of isoliensinine, neferine in plasma was determined using a calibration curve for each analytical batch. Pharmacokinetic parameters include elimination half-life (t 1/2 ) Area under concentration-time curve (AUC), mean Residence Time (MRT), apparent distribution volume (V) d ) And total plasma Clearance (CL), calculated by non-compartmental model of DAS (drug and statistics, version 2.0) software.
2.7 results and conclusions
Results: the main pharmacokinetic parameters of isoliensinine, neferine in mice were obtained by pharmacokinetic analysis (table 3). The results show that the isoliensinine exhibits better pharmacokinetic parameters compared with the neferine. Conclusion: compared with the methyl lotus plumule, the isoliensinine has better relative bioavailability and high safety, and provides a certain basis for early drug-forming evaluation of the isoliensinine and the methyl lotus plumule.
TABLE 3 Primary pharmacokinetic parameters of isoliensinine, neferine in mice (mean.+ -. Standard deviation)
Example 3 (in vitro experiments): in vitro culture of hepatic stellate cells, observation of inhibition of isoliensinine and neferine on hepatic stellate cell viability
3.1 cell lines
Human hepatic stellate cell line LX-2 and rat hepatic stellate cell line T6
3.2 drugs and agents
DMEM (Shanghai biosciences limited), fetal bovine serum (nanking Bei Ga biosciences limited), PBS (laboratory home-made), trypsin (bifrox, cat. 1004GR 100), EDTA (BIOSHAP), MTT (bi cloud).
3.3 instruments
Ultra clean bench (ESCO BIOTECH), CO 2 Incubator (Thermo Scientific), full-automatic autoclave (micro-zero, U.S.), ultra-low temperature refrigerator (Haier), pure water meter (south kyohan laboratory equipment limited), electronic balance (saidolis limited, germany), enzyme-labeled meter (Bio Tek).
3.4 Experimental methods
And detecting the cell viability of isoliensinine and neferine to LX-2 and T6 by adopting an MTT method. Log grown LX-2 and T6 cells were trypsinized, inoculated into 96 well plates, and incubated at 5000 cells/well to 60% cell density, and 5 multiplex wells were set at 5 multiplex wells per concentration with 200 μl of total cell system, with 0, 0.3, 0.63, 1.25, 2.5, 5, 10, 20, 40, 80, 160 μl of isoliensinine, or 0, 0.9375, 1.875, 3.75, 7.5, 15, 30, 60, 120, 240, 480 μl of neferine.
After 24h of completion of the reaction, 20. Mu.l of 5% strength thiazole blue (MTT) solution was added to each well and incubation was continued for 4h. The supernatant was then aspirated, 200 μl of DMSO was added to each well, and the mixture was shaken on a shaker for 10min at low speed to allow the crystals to dissolve well. The absorbance of each well was measured at 490nm on a microplate reader.
3.5 results and conclusions
Results: the isoliensinine inhibits the cell viability of LX-2 and T6 at 10 mu M, the inhibition is enhanced along with the increase of the dosage, and obvious dose dependence is presented; the cell viability of the neferine inhibiting LX-2 and T6 at 30 μm was enhanced with increasing dose, showing a clear dose dependence (fig. 2, 3).
Conclusion: the isoliensinine and the neferine have the effect of inhibiting hepatic stellate cell activity in vitro, and the action effect of the isoliensinine is superior to that of the neferine.
Example 4 (in vitro experiments): in vitro culture of hepatic stellate cells, observation of the action of isoliensinine and neferine on hepatic stellate cell activation index mRNA expression
4.1 cell lines
Human hepatic stellate cell line LX-2 and rat hepatic stellate cell line T6
4.2 drugs and agents
DMEM (shanghai source biotechnology Co., ltd.), fetal bovine serum (Nanjing Bei Ga biotechnology Co., ltd.), PBS (laboratory self-made), trypsin (BIOFROX, cat. 1004GR 100), EDTA (BIOSHAP), trizol (ambion), chloroform (national medicine Convergence chemical Co., ltd.), isopropanol (Nanjing chemical Co., ltd.), 75% ethanol (laboratory self-made), DEPC water (Beyotime), reverse transcription kit (shanghai Santa biotechnology Co., ltd., product No. 11141ES 60), amplification kit (Shanghai Santa biotechnology Co., ltd., product No. 11201ES 60).
4.3 instruments
Ultra clean bench (ESCO BIOTECH), CO 2 Incubator (Thermo Scientific), full-automatic autoclave (micro ZEALWAY, USA), ultra-low temperature refrigerator (Haier), pure water meter (Nanj Hanlong laboratory Equipment Co., ltd.), refrigerator (Haier), electronic balance (Sidoris Limited, germany), real-time fluorescent quantitative PCR instrument (Bio-Rad), nanodrop One (ThermoFisher).
4.4 Experimental methods
Taking LX-2 and T6 cells growing logarithmically, digesting the cells by trypsin, inoculating the cells into a 10cm dishWhen the density reaches 60%, after the isoliensinine (5, 10, 20 mu M) or the methyl liensinine (15, 30, 60 mu M) acts for 24 hours, 1mL of Trizol is added, the mixture is blown and collected, and the mixture is kept stand at room temperature for 5 minutes and centrifuged for 15 minutes at the temperature of 4 ℃ and 12000 rmp/min; centrifuging, transferring the colorless water phase at the upper layer of 0.5mL into a new enzyme-free EP tube, adding 0.5mL of isopropanol, mixing uniformly, standing for 10min, and centrifuging at 4 ℃ for 12000rmp/min for 10min; centrifuging to remove supernatant, collecting white precipitate as RNA, adding 1mL of 75% ethanol into each hole, shaking to suspend RNA precipitate, centrifuging at 4deg.C and 7500rmp/min for 5min, sucking liquid with gun head, drying in fume hood for 15min, adding 20 μL of DEPC water, dissolving to obtain mRNA of cell, measuring concentration and purity with Nanodrop, and determining the concentration and purity according to the following stepsII 1st Strand cDNA Synthesis kit reagent operation shows that the reverse transcription system is prepared to obtain cDNA of tissue and cell. The cDNA obtained was according toqPCR SYBR Green Master Mix kit reagent operation instructions for preparing an amplification system, performing an amplification procedure according to the instructions to obtain a gene amplification CT value, normalizing the result with rat GAPDH or human GAPDH, and using 2 -ΔΔCT The calculation method performs data statistical analysis.
4.5 results and conclusions
Results: isoliensinine and neferine can reduce mRNA expression levels of activation index alpha-SMA and COL1 alpha 1 genes in hepatic stellate cells in a dose-dependent manner, and the isoliensinine has a significant difference (P < 0.01) at low doses and the neferine has a significant difference (P < 0.01) at high doses (figures 4 and 5).
Conclusion: the isoliensinine and the neferine exert the effect of inhibiting the activation of hepatic stellate cells in vitro, and the effect of the isoliensinine is superior to that of the neferine.
Example 5 (in vitro experiments): and culturing hepatic stellate cells in vitro, and observing the effect of isoliensinine and neferine on hepatic stellate cell activation index protein expression.
5.1 cell lines
Human hepatic stellate cell line LX-2 and rat hepatic stellate cell line T6
5.2 drugs and agents
DMEM (Shanghai source culture biosciences limited), fetal bovine serum (Nanjing Bei Ga biosciences limited), PBS (laboratory home control), trypsin (BIOFROX, cat. 1004GR 100), EDTA (BIOSHAP), lysate (Biyun), electrophoretic fluid (laboratory home control), exposure fluid (Nanjing goodbye limited), PVDF membrane (GVS North America Sanford, ME).
5.3 instruments
Ultra clean bench (ESCO BIOTECH), CO 2 Incubator (Thermo Scientific), full-automatic autoclave (micro ZEALWAY, USA), ultra-low temperature refrigerator (Haier), pure water meter (Nanj Hanlong laboratory instruments Co., ltd.), refrigerator (Haier), electronic balance (Sidoris Co., germany), exposure meter (Bio-Rad).
5.4 Experimental methods
Log grown LX-2 and T6 cells were trypsinized, inoculated into 10cm dishes, and when the cell density reached 60%, after 24h of action with isoliensinine (5, 10, 20 μm) or methylliensinine (15, 30, 60 μm), lysed with RIPA lysis buffer, and cell lysates were collected and stored overnight at-20 ℃. The protein mixture was thawed, centrifuged at 12000rpm at 4℃for 15min, and the supernatant was aspirated and the protein concentration was measured by BCA method. Protein loading amount is 40 mug, 12% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) electrophoresis is carried out, PVDF transfer film is carried out, and nonspecific blocking is carried out; the corresponding primary antibody was added overnight at 4℃and horseradish peroxidase-labeled secondary antibody was added for hybridization. ECL luminescence was added and imaged and analyzed with a gel imager (Bio-Rad).
5.5 results and conclusions
Results: isoliensinine and neferine can reduce mRNA expression levels of activation indexes COL1A1 and ACTA2 genes in hepatic stellate cells in a dose-dependent manner, and the isoliensinine has a significant difference (P < 0.01) at a low dose, and the neferine has a significant difference (P < 0.01) at a high dose (FIGS. 6 and 7).
Conclusion: the isoliensinine and the neferine exert the effect of inhibiting the activation of hepatic stellate cells in vitro, and the effect of the isoliensinine is superior to that of the neferine.
Example 6 (in vitro experiments): and (3) culturing hepatic stellate cells in vitro, and observing the effect of isoliensinine and neferine on hepatic stellate cell death.
6.1 cell lines
Human hepatic stellate cell line LX-2 and rat hepatic stellate cell line T6
6.2 drugs and Agents
DMEM (Shanghai source biosciences limited), fetal bovine serum (nanking Bei Ga biosciences limited), PBS (laboratory home-made), trypsin (bifrox, cat No. 1004GR 100), EDTA (bishap), trypan blue kit (bi cloud).
6.3 instruments
Ultra clean bench (ESCO BIOTECH), CO 2 Incubator (Thermo Scientific), full-automatic autoclave (micro ZEALWAY, USA), ultra-low temperature refrigerator (Haier), pure water meter (Nanj Hanlong laboratory instruments Co., ltd.), refrigerator (Haier), electronic balance (Sidoris Limited, germany), microscope (ZEISS).
6.4 Experimental methods
Log grown LX-2 and T6 cells were trypsinized, inoculated into six well plates, and when the cell density reached 60%, the cells were digested with EDTA-free trypsin and collected by centrifugation after 24h of action with isoliensinine (5, 10, 20 μm) or methylliensinine (15, 30, 60 μm), and then the collected cells were washed twice with PBS and resuspended. mu.L of resuspended cells were pipetted into a plastic centrifuge tube, 10. Mu.L of trypan blue staining solution was added, gently mixed and stained for 1min. The cells were counted with a cytometer and images were acquired in random fields. Stained blue cells represent dead cells.
6.5 results and conclusions
Results: trypan blue results showed that isoliensinine, neferine, induced activated hepatic stellate cell death dose-dependently (fig. 8).
Conclusion: the isoliensinine and the neferine exert the effect of inducing the death of the activated hepatic stellate cells in vitro, and the effect of the isoliensinine is superior to that of the neferine.
While the preferred embodiments of the present invention have been illustrated and described, the present invention is not limited to the embodiments, and various equivalent modifications and substitutions can be made by one skilled in the art without departing from the spirit of the present invention, and these equivalent modifications and substitutions are intended to be included in the scope of the present invention as defined in the appended claims.
Claims (4)
1. The application of isoliensinine in preparing medicine for treating liver fibrosis is provided.
2. The application of isoliensinine in preparing anti-liver fibrosis medicine according to claim 1, wherein the anti-liver fibrosis medicine is a pharmaceutical composition composed of active component isoliensinine and pharmaceutically acceptable auxiliary materials.
3. The use of isoliensinine according to claim 1 or 2 for the preparation of an anti-liver fibrosis medicament, wherein the active ingredient isoliensinine in the pharmaceutical composition is 0.1% -99.9% of the total weight.
4. The use of isoliensinine according to claim 1 or 2 for the preparation of an anti-liver fibrosis medicament, wherein the pharmaceutical composition is in the form of a tablet, granule, pill, capsule or injection.
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| 莲子心对肝纤维化大鼠的抗脂质过氧化作用研究;施京红等;《中药材》;第39卷(第8期);第1869-1872页 * |
| 莲子心醇提物抗四氯化碳诱导大鼠肝纤维化的实验研究;高天娇等;《中国中西医结合杂志》;第34卷(第12期);第1476-1480页 * |
| 莲子的养生保健作用;单玉刚;《中国药膳研究会首届药膳大会》;第368-371页 * |
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