CN110755621B - A composition comprising Senp expression regulating reagent and its application in the preparation of anti-hepatic fibrosis drugs - Google Patents
A composition comprising Senp expression regulating reagent and its application in the preparation of anti-hepatic fibrosis drugs Download PDFInfo
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- CN110755621B CN110755621B CN201911147048.4A CN201911147048A CN110755621B CN 110755621 B CN110755621 B CN 110755621B CN 201911147048 A CN201911147048 A CN 201911147048A CN 110755621 B CN110755621 B CN 110755621B
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Abstract
The invention discloses a composition containing a Senp expression regulating reagent and an FXR agonist, and application of the composition in preparation of anti-hepatic fibrosis drugs. Compared with the prior art, the test result of the invention shows that the combination of the Senp expression regulating reagent and the FXR agonist can up-regulate the expression of Senp7, effectively reduce the SUMO level of FXR protein, improve the FXR transcription activity and have the obviously improved anti-hepatic fibrosis effect.
Description
Technical Field
The invention belongs to the technical field of anti-hepatic fibrosis medicines, and particularly relates to an anti-hepatic fibrosis composition and application thereof.
Background
Liver diseases seriously harm human health and have extremely high incidence rate worldwide, wherein the occurrence and development of liver diseases such as viral hepatitis, fatty liver disease, liver cancer and the like take the development of liver fibrosis as a pathological basic link. Liver Fibrosis (Fibrosis) is a pathological condition, which refers to the excessive deposition of fibrous connective tissue in the liver. Hepatic fibrosis is an intermediate link of further development of chronic liver diseases such as chronic viral hepatitis, metabolic disorder, chronic alcoholic/non-alcoholic fatty liver and the like to liver cirrhosis, the hepatic fibrosis is a common important characteristic of chronic liver diseases, and 25-40% of patients with chronic liver diseases finally develop liver cirrhosis and even liver cancer. Therefore, the key link for inhibiting the occurrence and the development of chronic liver diseases is to inhibit the formation of hepatic fibrosis. The site of Hepatic fibrosis is concentrated in Hepatic Stellate Cells (HSCs) in the liver, and when the liver is damaged, cytokines produced by the cell interaction between the Hepatic Stellate Cells and the Hepatic parenchymal Cells and Kupffer Cells induce the self-activation of the Hepatic Stellate Cells, activate the extracellular matrix (ECM) produced by secretion, cause the deposition of collagen in the liver, and secrete a large amount of pro-fibrotic factors, thereby causing the formation of fibrosis. Therefore, HSCs are the determinants of the development of liver fibrosis, and inhibition of HSCs activation is an important strategy for the treatment and reversal of liver fibrosis.
The research and development of anti-hepatic fibrosis drugs are the research and development hotspots of the current liver drugs, and meanwhile, certain research progress is also made. The currently reported therapeutic drugs for preventing and treating hepatic fibrosis are mainly in the following categories: 1) chinese herbal medicine and its extract, such as Curcuma rhizome, resveratrol, silibinin, allocryptopine and its salt (grant No. CN 10132721B); 2) chemical agents and formulations thereof, such as pirfenidone and creatinine combinations (grant publication No. CN103550242B), Cleistanone derivatives (grant publication No. CN 104095857B); 3) biological agents, including recombinant proteins and gene drugs, etc., the former including TGF beta 1-inhibitory peptide (grant No. CN1203091C), IL-4 (grant No. CN101318013B), monoclonal antibody HAb18GC2 and its heavy and light chain variable degene and polypeptide (grant No. CN 100586960C); the gene medicine includes hepatocyte nuclear factor 1 alpha gene (grant No. CN102552935B), gene medicine expressing hepatocyte poietin (application No. 200610145523.0), human hepatocyte growth factor gene (grant No. CN1142272C) and the like. Among them, Farnesoid X Receptor drugs have great research prospects, and Farnesoid X Receptor (FXR) is also called NR1H4(Nuclear Receptor subset 1, Group H, Member 4), which is a Member of Nuclear Receptor superfamily. Since its cloning in 1995, the receptor has become increasingly functionally recognized: FXR plays an important role in physiological processes such as bile acid, lipid and carbohydrate metabolism and has a regulation and control effect on various pathological processes. In addition, obeticholic Acid (also known as OCA) has been approved by the U.S. FDA to market as a potent FXR agonist in 2016 (Markham A, Keam SJ. Obeticholic Acid: First Global application. drugs.2016 Aug; 76(12):1221-6.) in 5 months as an anti-Primary Biliary Cirrhosis (PBC) drug, which has been the First drug to successfully market with FXR as a target. Although results of a number of previous animal experiments indicate that FXR agonists such as OCA have significant therapeutic effects on liver fibrosis (Fiorucci S, Antonelli E, Rizzo G, et al. the nuclear receptor SHP media inhibition of hepatic fibrous cells by FXR and protection against infection liver. gastroenterology.2004 Nov; 127(5) 1497-512.Fiorucci S1, Rizzo G, Antonelli E, et al. A facial receptor-small liver receptor regulation of fibrous tissue inhibition of viral infection inhibition primer inhibition-1 and matrix inhibition in liver fibrosis of liver tissue 584 J.95. 2. the FXR agonists such as OCA and OCA have significant therapeutic effects on liver fibrosis and liver fibrosis in liver fibrosis of liver fibrosis J.314. the experimental results of mouse theory and liver fibrosis J.95. the FXR inhibitors of liver cancer cells 584. 95. the FXR 1. the FXR inhibitors of OCA. the FXR agonists of OCA. the FXR and liver cells of the FXR agonists of the OCA. 1. the FXR and the FXR agonists of the FXR inhibitors of liver fibrosis of the FXR and liver cells of the same origin of the same No. 95. 3. 1. the FXR; however, a recent clinical study showed that OCA did not significantly improve the liver fibrosis index of PBC patients compared to Placebo (Nevens F, Andreone P, Mazzella G, et al. A Placebo-Controlled Trial of Obeticholic Acid in Primary Biliary Cholingitis. N Engl J Med.2016 Aug 18; 375(7):631-43.), and phase III clinical Trial of Obeticholic Acid anti-NASH completed in 2019 by Intercept corporation showed that Obeticholic Acid improved liver fibrosis symptoms in 23% of patients (Placebo 12%), the drug effect was only 11% higher despite the statistical difference, and the drug effect was not as satisfactory as expected. Although FXR agonists represented by obeticholic acid are expected to be the first approved drugs for improving hepatic fibrosis symptoms associated with fatty liver, the phenomenon that the effect of FXR agonists on developed hepatic fibrosis is not obvious is not explained and solved. Research and discovery of methods for improving the anti-fibrotic effects of FXR agonists have also become an undeniable focus of research.
SUMO (small ubiquitin related-modetier) protein widely exists in eukaryotic animal bodies, is a polypeptide consisting of 98 amino acids, and plays an important regulation role in the bodies. The SUMO modification is based on the covalent binding of SUMO as a structural basis, and combines with amino acid residues of a target protein; the SENP family is a protease for specifically degrading intracellular SUMO modified proteins, and is widely involved in the SUMO degradation process. In the present study, it was found that direct termination of SUMO process by lysine binding site mutation can greatly improve the transcriptional activity of calpain (Yang S H, Jaffray E, Hay R T, et al. dynamic interaction of the SUMO and ERK pathway in regulating Elk-1transcriptional activity [ J ] Molecular Cell,2003,12(1):63-74.), and the transcriptional activity of HDAC1 protein over-expressing SUMO-specific protease (SENP, Sentrin/SUMO-specific proteases) family is also improved, while the involvement of SENP significantly reduces the HDAC1-SUMO level at this time.
In previous studies, applicants found that the increase of SUMO level of FXR protein in activated hepatic stellate cells is the cause of weakening or even disappearance of FXR ligand biological activity (patent application No. 201811534024.X), and proposed the application of a combination of a SUMO inhibitor and an FXR agonist in preparing anti-hepatic fibrosis drugs, wherein the SUMO inhibitor can significantly inhibit SUMO polypeptide from binding to FXR protein, so that the anti-fibrotic pharmacological activity of the combination is significantly improved. However, the above composition has a limitation that SUMO inhibitors can block SUMO from binding to FXR protein only, but have no effect on the protein to which SUMO has been bound. However, hepatic stellate cells of patients with hepatic fibrosis are in an activated state and FXR protein has bound SUMO. Then for FXR proteins that have bound SUMO, further strategies need to be found to degrade the SUMO polypeptide and thereby increase the biological activity of FXR agonists.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the technical problems, the invention provides a composition for resisting hepatic fibrosis and application thereof, wherein the composition comprises a Senp expression regulating reagent and an FXR agonist, and tests prove that the combination of the Senp expression regulating reagent and the FXR agonist has a remarkably improved hepatic fibrosis resisting effect.
The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a composition for anti-liver fibrosis, comprising a Senp expression modulating agent and an FXR agonist.
Preferably, the method comprises the following steps:
the Senp expression regulating agent is various chemical, natural products or biological products and the like capable of inducing (up-regulating) the expression of Senp7 or blocking the degradation of Senp7 protein, and comprises the following components:
vectors carrying the Senp7 plasmid and capable of interfering with the expression of Senp7, which can up-regulate the expression of Senp7 (mRNA level and protein level) after transfecting cells or organisms, for example, viral vectors (lentiviruses, adenoviruses and the like), non-viral vectors (cationic polymer vectors, nanoparticle vectors, polyethyleneimine, liposomes, biocompatible carrier materials), and various vectors obtained by modifying the surface of the vectors with groups. Preferably, liposomes are representative of non-viral vectors in the present invention.
The FXR agonist is an agonist capable of promoting FXR transcriptional activation, and comprises FXR agonists of natural origin, semi-synthetic and full chemical synthesis. For example, selected from, but not limited to, obeticholic acid.
The invention also provides application of the composition in preparing anti-hepatic fibrosis drugs.
The invention also provides application of the composition in preparing a medicament for treating liver fibrosis diseases. Preferably, the hepatic fibrosis-associated disease is a liver disease such as fatty liver, drug-induced liver injury, or viral liver injury.
The invention also provides application of the composition in preparing a medicine for reducing the SUMO level of FXR protein in activated hepatic stellate cells.
The composition can be a medicine prepared by taking a Senp expression regulating reagent and an FXR agonist as active ingredients and adding pharmaceutically acceptable auxiliary materials.
The pharmaceutically acceptable auxiliary materials refer to various conventional auxiliary materials required when different dosage forms are prepared, such as diluents, adhesives, disintegrants, glidants, lubricants, flavoring agents, inclusion materials, adsorbing materials and the like, and the pharmaceutically acceptable auxiliary materials are prepared into any one of common oral preparations by a conventional preparation method, such as granules, powder, tablets, capsules, pills, oral liquid, decoction, dropping pills and the like.
The invention provides a composition containing a Senp expression regulating reagent and an FXR agonist, and application of the composition in preparing anti-hepatic fibrosis drugs. In a previous study by the applicant (patent application No. 201811534024.X), FXR agonists have no effect on the activation of hepatic stellate cells at all times in the highly activated state of hepatic stellate cells, due to SUMO modification of FXR protein in activated hepatic stellate cells; in addition, the applicant has also proposed in the previous project (patent application No. 201811534024.X) a composition of a SUMO inhibitor with an FXR agonist; the composition has effects in inhibiting hepatic stellate cell activation and treating hepatic fibrosis. However, the above scheme has certain limitations: namely, the SUMO inhibitor (including spectinomycin and/or ginkgolic acid and the like) has the action link of inhibiting the connection of the SUMO polypeptide to the modified protein, namely blocking the SUMO modification of FXR protein; but has no effect on FXR protein which is subjected to SUMO modification; however, most of the FXR protein has been modified by SUMO during hepatic stellate cell activation. Therefore, the above scheme has certain limitations. The Senp7 expression regulating reagent provided by the invention acts on the protein which is subjected to SUMO modification, and the SUMO polypeptide is hydrolyzed to obtain unmodified FXR protein. Therefore, aiming at the limitations of the previous scheme, the invention further provides a composition containing a Senp7 expression regulating agent and an FXR agonist, and the application of the composition in preparing an anti-hepatic fibrosis drug: the activated hepatic stellate cells have good reactivity to the compound, and the compound can obviously inhibit the activation of the hepatic stellate cells in an activated state, thereby obviously inhibiting hepatic fibrosis. Therefore, for hepatic fibrosis patients needing drug administration, hepatic stellate cells in the liver are already in an activated state, and the FXR protein is already subjected to SUMO modification; the combined use of the Senp7 expression regulating reagent and the FXR agonist can effectively reduce the SUMO level of FXR protein, improve the FXR transcription activity and enhance the effect of inhibiting the activation of hepatic stellate cells.
The technical effects are as follows: compared with the prior art, the invention provides a composition containing a Senp expression regulating reagent and an FXR agonist and application of the composition in preparation of anti-hepatic fibrosis medicines, and test results show that the combination of the Senp expression regulating reagent and the FXR agonist can up-regulate Senp7 expression, effectively reduce the SUMO level of FXR protein, improve FXR transcription activity and have a remarkably improved anti-hepatic fibrosis effect.
Drawings
FIG. 1 is a schematic representation of the functional blocks of SUMO inhibitors and Senp expression modulating agents.
FIG. 2 is an analysis of the mRNA expression level of Senp in activated HSCs. P <0.05 compared to resting HSCs.
FIG. 3 shows the analysis of the expression level of Senp7 mRNA after HSCs are transfected with the expression plasmid of Senp 7. P <0.001 was compared to the transfection control plasmid.
FIG. 4 shows the SUMO level analysis of FXR protein after HSCs are transfected with Senp7 expression plasmid. P <0.001 was compared to the transfection control plasmid.
FIG. 5 shows the regulation effect (Shp mRNA level) of FXR target gene by the combination of the Senp7 expression plasmid and OCA. P <0.05 compared to control group.
FIG. 6 shows the effect of the combination of the Senp7 expression plasmid and OCA on HSC activation (Acta2 mRNA levels). P <0.05 compared to control group.
Detailed Description
In previous studies, the applicant finds that the SUMO level of FXR protein in HSCs in an activated state is obviously higher than that in a resting state by separating and comparing HSCs in the resting state and the activated state; and results in the failure of the FXR agonist OCA to inhibit HSCs cell activation. In the research of the invention, the composition of the Senp expression regulating reagent and the FXR agonist is provided, and the composition can obviously reduce the SUMO level of FXR in activated HSCs, improve the transcriptional activity of FXR and inhibit the activation of the HSCs.
Specifically, in activated HSCs cultured in vitro, the SUMO-specific protease family responsible for degradation of intracellular SUMO proteins was investigated and found to have significantly reduced expression of Senp 7. After the Senp7 plasmid is transfected to cause overexpression, SUMO protein degradation in activated HSC cells is increased, and SUMO level of FXR protein is remarkably reduced. The combination of the Senp7 expression reagent and the obeticholic acid OCA can obviously enhance the regulation and control effect on FXR transcriptional activity and obviously up-regulate the target gene level of Shp; and obviously inhibits the activation of HSCs and the expression of Acta2 gene.
The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The biological materials (experimental animals, plasmids, adenoviruses, etc.) and reagents used in the examples of the present invention are commercially available, unless otherwise specified.
Example 1 decreased Effect of FXR agonist obeticholic acid administration during HSCs activation
1 materials of the experiment
The experimental mouse (C57BL/6) used in the invention is purchased from Beijing Wintolite laboratory animal technology GmbH;
collagenase type IV, Dnase E and Dnase I, Histodenz are all purchased from Sigma-Aldrich company, and the experimental buffer solution is prepared according to the formula of the standard buffer solution; obeticholic acid used in the present invention is available from MedChem, reverse transcription reagent from Vazyme, Trizol RNAisso plus from TAKARA.
The remaining reagents and materials are commercially available.
2 method of experiment
2.1 isolated culture of HSCs
After the mice are anesthetized to a proper degree by 5% chloral hydrate, abdominal disinfection is carried out, the mice are fixed on a foam board, then the mouse skin is fixed by a needle, the abdominal cavity of the mice is opened by a pair of scissors, and the abdominal cavity is exposed after the opening is in a V shape.
The large forceps are used for dragging the intestinal tract and the stomach to the right side of the body to expose the hepatic portal vein, the forceps are used for separating the hepatic portal vein carefully, the wall of the vein vessel is guaranteed to be moist and smooth, tissue mesentery adhesion on the vessel is guaranteed to the greatest extent, the contact pin is influenced, and meanwhile, two thread buries are arranged below the vessel.
Carefully support the vessel with the forceps handle and simultaneously insert the vessel slowly with the indwelling needle, about 1.5cm to 2.5cm, while finding backflow of the hepatic vessel into the indwelling needle.
At the moment, the metal needle head is immediately pulled out, the plastic needle is fixed on the hepatic portal vein by a previously buried line, the constant flow pump is started, the flow rate is adjusted to 12-15 ml/ml, the slight swelling and yellowing of the liver are observed, the inferior vena cava is immediately opened, and the blood flow is enabled to flow out quickly.
Keeping the position of the needle head relatively fixed, fully perfusing the blood of the liver by using a preheated D-hanks buffer solution, and observing the softening of the liver while perfusing, wherein when the color is deep and light, if poor perfusion exists, a sterile glove or a sterile cotton swab can be used for squeezing and massaging.
After 10min to 15min, the liver is basically paralyzed and the inferior vena cava bleeding basically disappears, perfusion is carried out by using prepared perfusion enzyme liquid, the flow rate is reduced to 8ml/min, no bubble can appear in the process and enters a pump tube, when the enzyme liquid fully digests liver tissues, cracked liver textures are observed, and the liver becomes paralyzed and softened (a cotton ball is used for absorbing a little liquid possibly flowing out from a blood vessel intubation part in the whole process, so that the condition that the blood vessel is not excessively filled to cause the falling of a needle head is ensured). Meanwhile, the whole liver was quickly separated with scissors into a small dish in ice bath, and soaked with hanks buffer solution for transfer.
Moving to a super clean bench and mechanically separating by using sterile tweezers. The shaken hepatocytes were dispersed in hanks buffer (50ml), added with the necessary digestive enzymes (pre-prepared in tubes) and transferred to a 50ml centrifuge tube and shaken vigorously for 40min at 37 ℃ in a shaker at 180rpm (this step must ensure high speed).
Taking out cells, wherein the cell suspension is muddy yellow, performing primary separation with a filter membrane (or gauze), removing undigested tissue, centrifuging the remained cell suspension at the rotating speed of 500rpm for 8min, and observing remarkable layering after centrifugation.
The precipitate was removed. And the operation is repeated once more. The precipitate was removed again to give a supernatant fluid predominantly of stromal cells, which was also yellowish in color but slightly lighter in color.
Centrifuging for 8min at 1750rpm by using a centrifuge, removing supernatant in the centrifuge tube after centrifugation is finished, re-suspending with hanks at 4 ℃, fixing the volume of all precipitates to 10ml, supplementing 20ml of Histodenz gradient centrifugate (sterile and prepared in situ) with the concentration of 18%, and fully suspending to obtain a mixed cell suspension with the total volume of 10+ 20-30 ml
And evenly distributing the mixed cell suspension into 4 15ml centrifuge tubes, wherein each centrifuge tube is 7.5ml, and continuously slowly inclining the upper hanks buffer solution, and each centrifuge tube is 2 ml. The process ensures that the density of the cell mixed liquid is not destroyed, and the process is gentle and slow.
And (3) continuing to centrifuge after further balancing, regulating the lifting speed to the lowest level, centrifuging for 20min at 3200rpm to obtain cell gradient liquid with a white turbid cell zone in the middle, slightly sucking by using a dropper, and properly sucking more cells, and then purifying by centrifuging.
Diluting the sucked turbid cell band with a proper amount of hanks, centrifuging at 1400rpm for 5min to obtain cell sediment, then resuspending and centrifuging with DMEM containing an anti-culture medium (several times of washing can be performed), and performing spot plate culture. After about 2h to 2.5h, the culture medium can be attached to the wall, and the culture medium is changed the next day. The number of culture days was counted from the next 24h day of culture time, and cells of d 1and d7 were collected and counted as hepatic stellate cells in the resting state and in the activated state.
2.2 RT-PCR
2.2.1 Total RNA extraction from hepatic stellate cells
1) Washing cells with PBS, adding 1ml Trizol reagent, homogenizing with a tissue homogenizer, standing at room temperature for 5min, and centrifuging at 12000g for 5 min;
2) the supernatant was transferred to 800. mu.l, 160. mu.l of chloroform was added thereto, the mixture was vigorously shaken for 15sec, and then centrifuged at 12000g for 15min after leaving at room temperature for 5 min. The sample was divided into three layers: the bottom layer is a yellow organic phase, and the upper layer is a colorless aqueous phase and an intermediate layer.
3) Carefully transferring 300 mu l of the upper-layer water phase into a new tube, adding 300 mu l of isopropanol, reversing, uniformly mixing, standing at room temperature for 10min, then centrifuging at 12000g for 10min, and discarding the supernatant;
4) the RNA pellet was washed with 1.0ml of pre-cooled 75% ethanol, then centrifuged at 12000g for 5min, the supernatant was discarded to give total RNA, which was redissolved with 30. mu.l of DEPC water, quantified and diluted to 0.5. mu.g/. mu.l.
2.2.2 reverse transcription
Preparing an RNA solution and kit components into a system with the total volume of 20 mul according to the system proportion required by the specification, setting a program temperature for reverse transcription, and specifically, the proportion is as follows:
2.2.3 PCR
the PCR system was as follows:
the PCR conditions were as follows:
stage 1: pre-denaturation at 95 ℃ for 1min
Stage 2: PCR reaction at 95 ℃ for 15 sec; such as 30sec for 40Cycles at 60 ℃; 72 ℃ 30sec
Stage 3: melting curve analysis at 65-95 deg.C, 0.5 deg.C/5 s
3 results of the experiment
3.1 after HSCs activation, obeticholic acid has reduced agonistic effect on FXR downstream target genes and reduced inhibitory effect on fibrotic genes
By inspecting the mRNA levels of the fibrosis gene and the FXR downstream target gene in a cell sample, the finding shows that the activated HSCs of obeticholic acid have weaker fibrosis drug effect, the downstream target gene has poorer agonistic effect, and the transcriptional activity is reduced.
3.2 FXR-SUMO level elevation after HSCs activation
According to the detection of an ELISA kit aiming at the SUMO level (figure two), the SUMO level of FXR protein in highly activated hepatic stellate cells after modeling is gradually increased along with the advance of culture time, and the SUMO level of the stellate intracellular protein in an activated state is higher than that in a resting state.
3.3 analysis of Senp expression levels after HSCs activation
According to the examination of six subtypes of mRNA of Senp family in HSCs after activation, the mRNA expression of Senp7 is reduced along with the activation of HSC cells (FIG. 2), suggesting that Senp7 is involved in the desSUMO during the activation of HSCs, and the reduction of the expression level of Senp7 leads to the increase of the SUMO level of protein in HSC cells in an activated state.
Example 2 the Senp7 overexpression plasmid can reduce SUMO level of activated HSC cells
1 materials of the experiment
the remaining experimental materials were the same as in example 1.
2 method of experiment
2.1 transfection of Senp7 plasmid
The Senp7 plasmid was transfected according to the Lipofectamine 2000 standard transfection method.
2.1.1 seeding of cells
Cells were seeded into twelve-well plates the day before transfection. The number of cells seeded depends on the growth rate, and is generally 1X 106And (4) cells. Cell confluence greater than 80% is required for transfection. Two hours before transfectionThe cells were exchanged to ensure transfection.
2.1.2 transfection plasmids
Plasmid DNA of the specified mass calculated in advance was added to a fixed volume of OPTI-MEM medium and mixed well. Simultaneously, a fixed volume of Lipo2000 was added to another equal volume of OPTI-MEM, gently mixed, and allowed to stand at room temperature for 5 minutes. The DNA suspension and the Lipo2000 suspension were then mixed together in a total volume of 250 ul/well (12 well plate), gently mixed and allowed to stand at room temperature for 20 minutes. And finally, adding the mixed solution of the DNA and the Lipo2000 into the holes of the cell plate, supplementing the culture medium, shaking the hole plate back and forth and left and right, mixing uniformly, and placing the mixture in an incubator for culture. After 4-6 hours of transfection, the cells were changed.
2.2 RT-PCR
The details are the same as 2.2 in example 1.
2.3 analysis of FXR protein SUMO levels in HSC cells
After extracting the nuclear protein according to the requirements of the ELISA detection kit specification, incubating the primary antibody and the nuclear protein of the cell sample in a plate hole for 60min at room temperature, supplementing and adding the SUMO specific detection antibody according to the requirements of the specification, further adding a color sensitizer for color development, then rapidly reading the light absorbance of 655nm, and carrying out relative quantitative analysis.
3 results of the experiment
3.1 verification of transfection efficiency of Senp7 expression plasmid in cells
Through the verification of the transfection effect of RT-PCR, the mRNA expression level of Senp7 in cells can be obviously improved after the cells are transfected with the Senp7 expression plasmid (figure 3).
3.2 analysis of the level of the FXR protein SUMO in HSCs after transfection of the Senp7 expression plasmid
SUMO levels of FXR protein were significantly reduced in activated hepatic stellate cells transfected with the Senp7 expression plasmid compared to the blank transfection group as measured by SUMO kit (fig. 4).
Example 3 Effect of the Senp7 expression plasmid in combination with OCA on hepatic stellate cell activation
1 materials of the experiment
The experimental materials were the same as in examples 1and 2.
2 method of experiment
2.1 Senp7 expression plasmid transfection
The transfection procedure for the Senp7 expression plasmid was the same as that described in section 2.1 of example 2.
Cells were transfected with Senp7 expression plasmid 48h, stimulated with 1 μ M OCA or equal volume of vehicle, and cells were harvested 12h later.
2.2 RT-PCR
The details are the same as 2.2 in example 1.
3 results of the experiment
3.1 study of FXR transcriptional Activity control Effect of Senp7 expression plasmid and OCA composition
HSC cells were transfected with the Senp7 expression plasmid, and after concurrent administration of OCA stimulation, HSC cells were examined for FXR transcriptional activity. The results show that the combination of the Senp7 expression plasmid and OCA can significantly increase the mRNA level of FXR target gene Shp in activated HSC cells (FIG. 5).
3.2 Studies on HSC cell activation by combination of Senp7 expression plasmid and OCA
HSC cells were transfected with the Senp7 expression plasmid, and after concurrent administration of OCA stimulation, HSC cells were examined for FXR transcriptional activity. The results show that the combination of the Senp7 expression plasmid and OCA can significantly increase the mRNA level of Acta2 in the HSC cells in the activated state (FIG. 6), and inhibit the activation level of HSC.
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