Resveratrol solid lipid nanoparticles and application of resveratrol solid lipid nanoparticles in preparation of drugs for treating hepatic fibrosis

Abstract

The invention discloses resveratrol solid lipid nanoparticles and application thereof in preparation of drugs for treating hepatic fibrosis, belongs to the technical field of drugs for treating hepatic fibrosis, and explains a regulation mechanism of the resveratrol solid lipid nanoparticles for resisting rat hepatic fibrosis in vivo and in vitro. Through CCK-8, cell scratches and Transwell experiments, the influence of the novel nano dosage form on the proliferation, migration and invasion ability of rat hepatic stellate cells (HSC-T6) is detected; an Annexin V-FITC / PI and ROS kit is used for determining the influence of the nano dosage form on the HSC-T6 apoptosis, the period and the ROS content; immunofluorescence and western-blot are used for detecting the influence of a new nano dosage form on the expression content of a hepatic fibrosis specific marker of HSC, and it is found that the resveratrol solid lipid nanoparticles can effectively inhibit proliferation of rat HSC-T6 cells, induce HSC-T6 cell apoptosis, inhibit migration and invasion and regulate alpha-SMA expression. The resveratrol solid lipid nanoparticles can inhibit the activation of hepatic stellate cells and achieve the effect of resisting rat hepatic fibrosis by influencing a signal channel of TGF-beta / smad2.

Description

Technical Field

[0001] The present invention belongs to the technical field of liver fibrosis drugs, and particularly relates to the application of resveratrol solid lipid nanoparticles in the preparation of drugs for treating liver fibrosis. Background Art

[0002] Liver fibrosis is one of the major liver diseases threatening human health and safety. The pathological process of liver fibrosis is complex, and various pathogenic factors, such as alcohol, obesity, drugs, trauma, hepatitis, etc., lead to abnormal proliferation of connective tissue in the liver. In the development of the pathogenesis of liver fibrosis, the cytological basis is the activation of hepatic stellate cells (HSCs). Currently, there is still a lack of effective chemical drugs for treating liver fibrosis clinically, and the treatment methods other than liver transplantation surgery are far from achieving the ideal treatment effect. Traditional Chinese medicine has great advantages and broad prospects in the treatment of liver fibrosis. Resveratrol (Res) has the effect of protecting the liver. Studies have shown that resveratrol exerts an anti-liver fibrosis effect by reducing oxidative stress responses. However, the effect of treating liver fibrosis with resveratrol solution alone is poor, mainly because resveratrol is difficult to enter the liver and has poor metabolic stability. The liver is an important organ for lipid metabolism and can recognize and uptake lipids through specific receptors. As those skilled in the art, there is an urgent need to develop a resveratrol solid lipid nanoparticle and its application in the preparation of drugs for treating liver fibrosis. Summary of the Invention

[0003] The purpose of the present invention is to provide a resveratrol solid lipid nanoparticle and its application in the preparation of drugs for treating liver fibrosis to solve the above technical problems.

[0004] Application of resveratrol solid lipid nanoparticles in the preparation of drugs for treating liver fibrosis, wherein the particle size distribution of the resveratrol solid lipid nanoparticles is 50 - 60 nm.

[0005] Further, the drug has at least one of the following characteristics: (1) inhibiting the activation of hepatic stellate cells; (2) affecting the TGF-β / smad2 signaling pathway.

[0006] Further, the drug contains at least resveratrol as an active ingredient.

[0007] Further, the drug contains resveratrol solid lipid nanoparticles.

[0008] Further, the drug contains one or more pharmaceutically acceptable carriers or adjuvants.

[0009] Further, the adjuvant is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

[0010] The above-mentioned resveratrol solid lipid nanoparticles are solid lipid nanoparticles encapsulating resveratrol, and the drug loading amount of resveratrol is 25.2%.

[0011] Further, the solid lipid nanoparticles encapsulating resveratrol are prepared by the emulsion evaporation-low temperature curing method. Specifically, the solid lipid and resveratrol are dissolved in an organic solvent at a temperature higher than the melting point of the lipid to form an organic phase; then the emulsifier is dissolved in distilled water at the same temperature to form an aqueous phase; after the organic solvent is removed by evaporation under mechanical stirring, the mixture is quickly dispersed in ice water and stirred for curing to obtain the product.

[0012] Resveratrol is a phenolic phytoalexin found in grape skins and other plants.

[0013] A pharmaceutical composition for treating liver fibrosis, characterized in that the pharmaceutical composition comprises the resveratrol solid lipid nanoparticles as described in claim 1.

[0014] Further, the above-mentioned pharmaceutical composition further contains one or more pharmaceutically acceptable carriers or adjuvants.

[0015] The present invention is implemented as follows: Liver fibrosis and cirrhosis are the main causes of morbidity and mortality of chronic liver diseases. The traditional Chinese medicine resveratrol has shown certain efficacy in inhibiting liver fibrosis. However, as a natural medicine, it has corresponding disadvantages in terms of solubility, bioavailability, etc. First, the drug dosage form of resveratrol is modified based on solid lipid nanoparticles. Compared with single-drug resveratrol, resveratrol solid lipid nanoparticles can more significantly inhibit the proliferation, apoptosis, and cell migration and invasion abilities of HSC-T6 cells.

[0016] Research shows that the PI3k / Akt signaling pathway plays an extremely important role in liver fibrosis. It participates in regulating various cell functions such as cell proliferation, transport, differentiation, and apoptosis. TGF-β / Smad regulates the activation of hepatic stellate cells and plays an important role in the occurrence and development of liver fibrosis. TGF-β II binds to TGF-β as a serine- and glycine-rich receptor and phosphorylates Smad2 / Smad3. The formed heterotrimer transports the signal from the cell membrane to the nucleus and participates in regulating a series of biological reactions.

[0017] Resveratrol solid lipid nanoparticles can inhibit the activation of HSC-T6 cells and improve the anti-fibrotic treatment effect by affecting the TGF-β1 / smad signaling pathway. It is expected to become a new strategy for the treatment of liver fibrosis. This also provides a reference for the treatment direction of using nano-formulations to carry natural drugs for the treatment of liver fibrosis.

[0018] The present invention has the following beneficial effects: By exploring the activation of hepatic stellate cells and the anti-rat liver fibrosis effect of resveratrol solid lipid nanoparticles (Res-SLNs), the regulatory mechanism of resveratrol solid lipid nanoparticles against rat liver fibrosis was preliminarily elucidated in vitro and in vivo. Resveratrol-solid lipid nanoparticles were prepared, and their morphology, charge, and phase were characterized and analyzed by transmission electron microscopy, X-ray diffraction, and infrared spectroscopy; CCK-8, cell scratch, and Transwell assays were used to detect the effects of the new nano-formulation on the proliferation, migration, and invasion abilities of rat hepatic stellate cells (HSC-T6); Annexin V-FITC / PI and ROS kits were used to determine the effects of the nano-formulation on the apoptosis, cell cycle, and ROS content of HSC-T6; immunofluorescence and western-blot were used to detect the effects of the new nano-formulation on the expression levels of liver fibrosis-specific markers in HSC, and its mechanism was further explored. It was unexpectedly found that resveratrol solid lipid nanoparticles can effectively inhibit the proliferation of rat HSC-T6 cells, and can also induce apoptosis of HSC-T6 cells, inhibit migration and invasion, and regulate the expression of α-SMA. Resveratrol solid lipid nanoparticles can be used in the preparation of drugs for the treatment of liver fibrosis. Description of the Drawings

[0019] Figure 1 A and Figure 1 B are transmission electron microscope pictures of SLNs and Res-SLNs; Figure 1 C and Figure 1 D are the zeta potentials of Res and Res-SLNs; Figure 1 E is the FTIR infrared spectrum of Res, SLNs, and Res-SLNs; Figure 1 F is the X-ray diffraction pattern of Res, SLNs, and Res-SLNs; Figure 2 are the graphs showing the effects of each treatment group on the proliferation, migration, and invasion abilities of HSC-T6 cells, where Figure 2 A is the detection of cell proliferation by CCK-8 method; Figure 2 B, Figure 2 C, Figure 2 D are the graphs of scratch and Transwell assays for detecting migration and invasion; Figure 3A are the graphs of cell apoptosis rates after different treatment groups act on HSC-T6 cells for 24 h, Figure 3B and Figure 3CIt is the cell cycle and ratio diagram after different treatment groups act on HSC-T6. Figure 3D It is the content of reactive oxygen species in HSC-T6 cells after different treatment groups act on HSC-T6 cells for 24 hours. Figure 4 It is the diagram of the influence of different administration groups on the expression content of the activation markers α-SMA and TGF-β in HSC-T6 cells detected by immunofluorescence. Figure 5 It is the protein expression level diagram of α-SMA, TGF-β1, Smad2 / 3, p-Smad2 / 3, PI3K, AKT, and p-AKT after different treatment groups treat HSC-T6 cells. Figure 6 It is the pathological section diagram of liver morphology observation of each treatment group and hematoxylin-eosin (H&E) staining of liver tissue sections. Specific implementation manners

[0020] The following uses specific examples to illustrate the present invention, but it does not limit the present invention.

[0021] The following reagents, cells, and animal sources are used: Resveratrol is purchased from Aladdin Chemical Reagent Company; Stearic acid, Lecithin, Chloroform, Myrj52 (Polyoxyethylene Stearate), SDS, Tween20 reagent, Methanol, Tissue Fixative are purchased from Sinopharm Chemical Reagent; DMEM, Fetal Bovine Serum (Hyclone company); Antibodies such as α-SMA, TGF-β1 (ABclonal company); CCK-8 kit, DAPI reagent, Annexin V&PI apoptosis and cycle kit, BCA kit, Western Blot gel preparation kit, Protein loading buffer (5×) (Beyotime company); Horseradish peroxidase-labeled rabbit anti-mouse (Nanjing Jiancheng Technology); Rat hepatic stellate HSC-T6 cell line is purchased from Procell company and cultured in DMEM containing 10% (v / v) fetal bovine serum at 37 °C under 5% CO2 saturated humidity conditions. SD rats, 180 g ± 20 g, male, clean grade, a total of 20 (Jinan Pengyue Experimental Animal Breeding Co., Ltd.), are raised in the Scientific Research Animal Center of Bengbu Medical College, and the breeding environment temperature is 22 - 25 °C, and the humidity is 55% - 60%. The animal disposal method ensures compliance with animal ethics requirements. Example 1

[0022] Morphology, potential, drug loading, X-ray crystal diffraction, and infrared characterization of SLNs, Res, and Res-SLNs.

[0023] Refer to the previous research work and prepare Res-SLNs by the "emulsion evaporation - low-temperature solidification" method. Prepare blank solid lipid nanoparticles SLNs according to the same steps (without resveratrol).

[0024] The brief steps are as follows: Resveratrol (150 mg), stearic acid (200 mg) and lecithin (100 mg) were dissolved in 10 mL of chloroform (using chloroform as the organic phase) and placed in a glass flask. Myrj52 was dissolved in 30 mL of distilled water and heated to 75 °C in a water bath (as the aqueous phase). The organic phase was injected into the hot aqueous phase under mechanical stirring at 1000 rpm, and the resulting solution was maintained at 75 °C at the same stirring speed to remove the organic solvent. Subsequently, 5 mL of the concentrated solvent was transferred to an equal volume of ice-cold water at 0 °C under continuous mechanical stirring (1000 rpm) and stirred for 2 hours. The resulting suspension was centrifuged at 20,000 rpm in an Avanti J25 centrifuge to remove the supernatant. The precipitate was washed with ultrapure water, refrigerated at -20 °C for 24 h, and then placed in a freeze dryer for 24 h.

[0025] As Figure 1 shown in Figures A and 1B, both SLNs and Res-SLNs nanoparticles were round, and the particle size distribution was between 50 - 60 nm. The potential was around -25 mV ( Figure 1 Figures C and Figure 1 D). The infrared spectrum of Res-SLNs had characteristic peaks of both solid lipid nanoparticles and resveratrol molecules, indicating that resveratrol was successfully encapsulated into the solid lipid nanoparticles ( Figure 1 Figure E). Res showed sharp peaks in the range of 5 - 20°, indicating a high crystalline structure, but these characteristics were not obvious in Res-SLNs, indicating that resveratrol embedded in the lipid core of SLNs was in an amorphous state ( Figure 1 Figure F). According to the standard curve for determining bound resveratrol measured by ultraviolet spectrophotometer, the drug loading of Res in Res-SLNs was 25.2%. Example 2

[0026] CCK-8, scratch and Transwell assays were used to detect the effects of different treatment groups on the proliferation, invasion and migration abilities of HSC-T6 cells.

[0027] Cell proliferation activity detection: The same concentration of SLNs, Res, and Res-SLNs were used to treat HSC-T6 cells for 24 hours, and the proliferation activity was determined using the CCK-8 kit. All experiments were performed at least 3 times. Migration ability: HSC-T6 was inoculated in a 6-well plate at a cell density of 5×105 cells per well, and the confluence was cultured to 90%. The cell monolayer was scratched with a 10μL pipette tip, and after washing and removing unattached cells, the cells were treated with SLNs, Res, and Res-SLNs for 24 hours and 48 hours, respectively. The migration ability of cells in different treatment groups at each time period was observed under an inverted microscope and photographed. At least 3 fields of view were observed for each group of cells. Complete culture medium was added to the 24-well plate, and the chamber was placed in the well; the drug-treated cells were collected and centrifuged, and divided into three groups: control group, Res-treated group, and Res-SLNs group. Incomplete culture medium was added to make a cell suspension, and the blood cell plate was counted 24 hours later; the chamber was taken out, air-dried, fixed and air-dried, and the dye was washed off after staining. After air drying, it was photographed and observed. Invasion experiment: After the matrix gel was spread in the Transwell chamber until the gel solidified, the number of cells entering the lower chamber was calculated to reflect the invasion ability of cells in different groups.

[0028] like Figure 2 As shown in A, compared with the SLNs and Res treatment groups, Res-SLNs can significantly inhibit the proliferation of HSC-T6 cells in a concentration-dependent manner, and can also significantly inhibit the migration of HSC-T6 cells ( Figure 2 B, Figure 2 D) and invasion ( Figure 2 C). Example 3

[0029] Flow cytometry was used to measure the changes in HSC-T6 cell apoptosis, cell cycle and reactive oxygen species (ROS) in different treatment groups.

[0030] Experimental groups: control group; resveratrol treatment group alone, material SLNs treatment group alone, Res-SLNs treatment group. HSC-T6 cells were inoculated in a six-well plate, the original culture medium was discarded after 24 hours, washed with PBS, and different treatment groups were added to each well at a final concentration of 50μMResveratrol (Res-SLNs group was converted according to the drug loading amount). After 24 hours of culture, the operation was carried out according to the instructions of the apoptosis kit. The cell cycle experimental grouping was the same as the apoptosis grouping, and the operation was carried out according to the cycle kit. ROS experiment: PBS was washed in each well, and the diluted DCFH-DA probe was added to each well, incubated at 37℃ for 30 minutes, DCFH-DA was discarded, and each well was washed three times with culture medium, and the cells were collected after trypsin digestion, and flow detection was performed in the dark.

[0031] like Figure 3A As shown, the apoptosis rate of HSC-T6 cells treated with Res-SLNs was significantly increased compared with that in the Res and SLNs groups.Figure 3D The ROS content in HSC-T6 cells increased, indicating that Res-SLNs promoted apoptosis more significantly. As Figure 3B and 3C shown, after treatment with Res-SLNs, the cell cycle of HSC-T6 cells was more significantly arrested in the G1 phase, and the proportions of the SLNs group and the control group were similar. Example 4

[0032] Immunofluorescence and Western-blot were used to detect the expressions of α-SMA, TGF-β, smad2 / 3, p-smad2 / 3, PI3K, and Akt.

[0033] Immunofluorescence: HSC-T6 cells were seeded in 24-well plates. After 24 h, the cells were treated with the working solutions of SLNs, Res, and Res-SLNs (50 μM), respectively. The cells were fixed, blocked with a blocking solution, incubated overnight at 4 °C, washed with PBS, and incubated with a secondary antibody dilution solution. DAPI working staining solution was added for incubation, and an anti-fluorescence quencher was added, followed by fluorescence inverted microscope photography and observation. Western-blot: After 24 h of cell seeding, the cells were treated with drugs for 24 h, washed with PBS, and collected. The cells were treated with RIPA lysis buffer for 30 min, centrifuged at 12,000 rpm for 10 min to collect the supernatant, and protein quantification was performed using a Bio-Rad kit. 8% SDS-PAGE was used for gel electrophoresis. The gel was transferred onto a PVDF membrane and blocked with 5% BSA at room temperature for 1 h. The membrane was mixed with the primary antibody according to the dilution ratio described in the instruction manual and incubated overnight at 4 °C. The membrane was washed 4 times with TBST, incubated with HRP-labeled goat anti-rabbit and rabbit anti-mouse secondary antibodies at 37 °C for 2 h, then washed, and the protein bands were analyzed and quantified by Image J software.

[0034] The activation of hepatic stellate cells is an important link in the occurrence and development of liver fibrosis, and α-SMA is the main marker of HSC activation. As Figure 4 shown, Res-SLNs could more significantly inhibit the expressions of α-SMA and TGF-β in HSC-T6 cells compared with the single drug and SLNs treatment groups. Example 5

[0035] Preparation of rat liver fibrosis model and drug administration protocol: First, establish the rat liver fibrosis model: 20 4-week-old SD rats were randomly divided into a normal control group, a control group, a Res administration group, and a Res-SLNs administration group, with 5 rats in each group. CCl4 injection was used to induce rat liver fibrosis modeling. Modeling grouping: Each group of rats was injected with a 20% CCl4 soybean oil solution by intraperitoneal injection. It was injected twice a week. Before each injection of CCl4, the body weight was weighed, and the dose was adjusted according to the increase or decrease in body weight. After successful modeling, drug administration began. The normal control group only provided normal diet and water without any other special treatment; the control group only modeled without drug administration. The Res group was administered at 10 mg / kg each time, and the Res-SLNs group was administered at a dose of 25 mg / kg each time after conversion according to the drug loading amount (equivalent to 10 mg / kg of resveratrol). The single drug was dissolved in DMSO below 1% and diluted with normal saline, and the combined drug was directly prepared with normal saline, and the rats were intraperitoneally injected (2 ml / kg). After four weeks of drug administration, the liver was taken and stored in 4% paraformaldehyde at room temperature. The liver tissue was sectioned with a thickness of 4 μm, stained with hematoxylin and eosin (H&E), to evaluate and determine the degree of liver lesions, and finally observed and photographed under a microscope.

[0036] The expression of key proteins in the TGF-β / Smad pathway and the PI3K / Akt signaling pathway in HSC-T6 cells of each group was detected by Western blot. As Figure 5 shown, compared with the CCl4 model group, the expression of α-SMA was significantly decreased in the Res and Res-SLNs treatment groups. Compared with the control group, Res-SLNs could more effectively inhibit the phosphorylation expression of TGF-β1, Smad2 / 3, Akt, and Smad3 to inhibit liver fibrosis. Example 6

[0037] Statistical processing: The data were statistically processed using SPSS 26.0 software. The experimental results were analyzed using the mean ± standard deviation (mean ± SD). The differences between groups were analyzed using a t-test. A P < 0.05 was considered to indicate a statistically significant difference.

[0038] As Figure 6 shown, the livers of the rats in the control group were bright red, smooth on the surface, soft in texture, and without a granular feeling. The livers of the rats in the CCl4 model group were dark red and yellowish, with a harder texture, a rough surface, and obvious granular feeling. The livers of the rats in the Res and Res-SLNs treatment groups were both dark red, slightly rough on the surface, but without obvious granular feeling. H&E staining showed that in the CCl4-treated model group, the liver structure was disordered, proliferated, fibrous connective tissue and inflammatory cells accumulated in the portal vein area. However, administration of Res and Res-SLNs could significantly improve the inflammatory infiltration, and the improvement effect of the Res-SLNs group was more obvious than that of the Res group.

[0039] In summary, liver fibrosis and cirrhosis are the main causes of morbidity and mortality in chronic liver diseases. The traditional Chinese medicine resveratrol has shown certain efficacy in inhibiting liver fibrosis. However, as a natural medicine, it has corresponding drawbacks in terms of solubility, bioavailability, etc. First, the drug formulation of resveratrol was modified based on solid lipid nanoparticles. Compared with single-drug resveratrol, resveratrol solid lipid nanoparticles can more significantly inhibit the proliferation, apoptosis, migration and invasion of HSC-T6 cells.

[0040] Studies have shown that the PI3k / Akt signaling pathway plays an extremely important role in liver fibrosis and is involved in regulating various cellular functions such as cell proliferation, transport, differentiation, and apoptosis. TGF-β / Smad regulates the activation of hepatic stellate cells and plays an important role in the development of liver fibrosis. TGF-βII binds to TGF-β as a serine- and glycine-rich receptor and phosphorylates Smad2 / Smad3. The resulting heterotrimer transports the signal from the cell membrane to the nucleus and participates in regulating a series of biological reactions.

[0041] Resveratrol solid lipid nanoparticles can inhibit the activation of HSC-T6 cells and improve the anti-fibrotic treatment effect by affecting the TGF-β1 / smad signaling pathway.

Claims

1. Use of resveratrol solid lipid nanoparticles in the preparation of a medicament for treating liver fibrosis.

2. Use of the resveratrol solid lipid nanoparticles according to claim 1 in the preparation of a medicament for treating liver fibrosis, characterized in that, The particle size distribution of the resveratrol solid lipid nanoparticles is 50 - 60 nm.

3. Use of the resveratrol solid lipid nanoparticles according to claim 1 in the preparation of a medicament for treating liver fibrosis, characterized in that, The medicament has at least one of the following properties: (1) inhibiting the activation of hepatic stellate cells; (2) affecting the TGF-β / smad2 signaling pathway.

4. Use of the resveratrol solid lipid nanoparticles according to claim 1 in the preparation of a medicament for treating liver fibrosis, characterized in that, The medicament contains resveratrol solid lipid nanoparticles.

5. A resveratrol solid lipid nanoparticle, characterized in that, The resveratrol solid lipid nanoparticles are solid lipid nanoparticles encapsulating resveratrol, and the drug loading of resveratrol is 25.2%.

6. The resveratrol solid lipid nanoparticle according to claim 6, characterized in that, The solid lipid nanoparticles encapsulating resveratrol are prepared by the emulsion evaporation - low temperature solidification method.

7. A pharmaceutical composition for preventing and treating liver fibrosis, characterized in that, The pharmaceutical composition includes the resveratrol solid lipid nanoparticles as described in claim 1.

8. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition further contains one or more pharmaceutically acceptable carriers or adjuvants.

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