Pharmaceutical composition containing mesenchymal stem cells for improving liver function

Abstract

The present invention discloses the use of mesenchymal stem cells and HP-16 polypeptide in preparing a drug kit for improving liver function, wherein the amino acid sequence of the HP-16 polypeptide is: RTIGVYKSHLDPEECM. Experimental results show that combined treatment with mesenchymal stem cells and HP-16 polypeptide significantly improves liver function and lipid metabolism indicators (P < 0.05), has a higher efficacy rate, and is significantly more effective in treating NAFLD than existing commonly used drug treatments. It can significantly improve liver function and metabolic status, and has good safety.

Description

Technical Field

[0001] The present invention relates to the technical field of biological stem cells, and in particular to a pharmaceutical composition containing mesenchymal stem cells for improving liver function. Background Art

[0002] The liver is the largest internal organ in the human body, performing multiple physiological functions, including metabolism, detoxification, storage, and secretion. Liver disease, including but not limited to hepatitis, cirrhosis, fatty liver disease, and liver cancer, is a significant global health concern. Traditional treatments for liver disease include medication, surgery, and liver transplantation. However, medications are limited in effectiveness, cannot reverse existing liver damage, and may cause adverse reactions. Surgical treatments, such as resection of damaged liver tissue, are high-risk and require a long recovery time. Liver transplantation is the only definitive solution for severe liver disease, but is limited by donor shortages, surgical risks, and post-transplant immune rejection.

[0003] Therefore, preventing and improving liver disease has become a key focus of attention, among which the development of safe, effective and sustainable strategies to improve liver function is even more important. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings of the prior art and provide a pharmaceutical composition containing mesenchymal stem cells for improving liver function. To achieve the above-mentioned object, the present invention provides the following technical solutions:

[0005] The present invention provides use of mesenchymal stem cells and HP-16 polypeptide in preparing a medicine kit for improving liver function. The amino acid sequence of the HP-16 polypeptide is: RTIGVYKSHLDPEECM.

[0006] Furthermore, the mesenchymal stem cells are isolated and cultured using the following method:

[0007] S1. Rinse fresh adipose tissue three times with PBS under sterile conditions to remove blood and impurities, and cut the adipose tissue into approximately 0.1*0.1cm 3 small pieces;

[0008] S2. Place the tissue fragment in a container containing 5 times the volume of collagenase type I and gently shake at 37°C to promote digestion for 40 minutes. Pipet with cell suspension every 10 minutes until the tissue is completely disintegrated.

[0009] S3. After digestion, add an equal volume of DMEM high-glucose medium containing 10% fetal bovine serum to stop digestion, filter through a 70-mesh filter, and then centrifuge at 1200 rpm for 5 minutes. Discard the supernatant, resuspend the cells in PBS and repeat washing three times to remove residual enzymes and impurities;

[0010] S4. The cell suspension was cultured in a culture dish containing homemade MSC culture medium, which consisted of 1000 mL of DMEM high glucose, 2.5 μg / L bFGF, 200 mL of KnockOut serum replacement, 10 mL of HyClone non-essential amino acids, and 10 mL of penicillin-streptomycin solution 100X. The culture was incubated at 37°C and 5% CO2.

[0011] S5. When the cells reach 80% confluence, digest them with 0.25% trypsin-EDTA solution for 2 min. Add an equal amount of DMEM high-glucose medium containing 10% fetal bovine serum to stop the digestion. Gently pipette to prepare a single-cell suspension. After centrifugation and washing, subculture the cells at a ratio of 1:3 to a new culture dish and continue culturing.

[0012] S6. When the cells reach passage P3, mesenchymal stem cells are harvested.

[0013] The present invention provides an HP-16 polypeptide, and the amino acid sequence of the HP-16 polypeptide is: RTIGVYKSHLDPEECM.

[0014] The present invention provides use of the HP-16 polypeptide in preparing a drug for improving liver function.

[0015] Furthermore, the HP-16 polypeptide is prepared by solid phase synthesis.

[0016] Furthermore, the medicine kit also contains a pharmaceutically acceptable carrier.

[0017] Furthermore, the carrier includes an excipient.

[0018] Furthermore, the medicine kit is also used together with a drug for improving liver function.

[0019] The present invention is different from the prior art in that the present invention achieves the following technical effects:

[0020] The present invention discovered through experiments that the experimental results show that the combined treatment of mesenchymal stem cells and HP-16 polypeptide has a significant improvement in liver function and lipid metabolism indicators (P<0.05), a higher effective rate, and a significantly better therapeutic effect for NAFLD than existing commonly used drug treatments. It can significantly improve liver function and metabolic status and has good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Comparison of liver function indicators between the control group and the treatment group of the present invention; wherein, subgraph A represents the ALP levels of the two groups of patients before and after treatment, subgraph B represents the ALT levels of the two groups of patients before and after treatment, and subgraph C represents the AST levels of the two groups of patients before and after treatment;

[0022] Figure 2 It is a comparison of the glucose and lipid metabolism indicators between the control group and the treatment group of the present invention; among them, sub-graph A shows the FBG levels of the two groups of patients before and after treatment, sub-graph B shows the TC levels of the two groups of patients before and after treatment, sub-graph C shows the LDL-C levels of the two groups of patients before and after treatment, and sub-graph D shows the HDL-C levels of the two groups of patients before and after treatment. DETAILED DESCRIPTION

[0023] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The materials and reagents used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0024] Example 1

[0025] The present invention discloses a method for isolating, culturing and preparing mesenchymal stem cells, which are prepared by the following method:

[0026] S1. Rinse fresh adipose tissue three times with PBS under sterile conditions to remove blood and impurities, and cut the adipose tissue into approximately 0.1*0.1cm 3 small pieces;

[0027] S2. Place the tissue fragment in a container containing 5 times the volume of collagenase type I and gently shake at 37°C to promote digestion for 40 minutes. Pipet with cell suspension every 10 minutes until the tissue is completely disintegrated.

[0028] S3. After digestion, add an equal volume of DMEM high-glucose medium containing 10% fetal bovine serum to stop digestion, filter through a 70-mesh filter, and then centrifuge at 1200 rpm for 5 minutes. Discard the supernatant, resuspend the cells in PBS and repeat washing three times to remove residual enzymes and impurities;

[0029] S4. The cell suspension was cultured in a culture dish containing homemade MSC culture medium, which consisted of 1000 mL of DMEM high glucose, 2.5 μg / L bFGF, 200 mL of KnockOut serum replacement, 10 mL of HyClone non-essential amino acids, and 10 mL of penicillin-streptomycin solution 100X. The culture was incubated at 37°C and 5% CO2.

[0030] S5. When the cells reach 80% confluence, digest them with 0.25% trypsin-EDTA solution for 2 min. Add an equal amount of DMEM high-glucose medium containing 10% fetal bovine serum to stop the digestion. Gently pipette to prepare a single-cell suspension. After centrifugation and washing, subculture the cells at a ratio of 1:3 to a new culture dish and continue culturing.

[0031] S6. When the cells reach passage P3, mesenchymal stem cells are harvested.

[0032] Example 2

[0033] The present invention discloses an HP-16 polypeptide having an amino acid sequence of: RTIGVYKSHLDPEECM (Arg-Thr-Ile-Gly-Val-Tyr-Lys-Ser-His-Leu-Asp-Pro-Glu-Glu-Cys-Met). The HP-16 polypeptide is prepared by solid-phase synthesis.

[0034] Example 3

[0035] 1. Research subjects and treatment methods

[0036] A total of 120 patients diagnosed with NAFLD were selected, with an age range of 18-65 years, including 82 males and 38 females, with an average age of 45.6±11.3 years.

[0037] Patients were divided into a control group and a treatment group based on the different treatment interventions they received, with 60 cases in each group. The control group received an oral treatment regimen involving a single drug: polyene phosphatidylcholine capsules; this drug is produced by Sanofi (Beijing) Pharmaceutical Co., Ltd., and each capsule contains 228 mg of polyene phosphatidylcholine; the initial treatment plan was to take two capsules orally three times a day (a total of 456 mg). After four weeks of treatment, the dosage was halved to one capsule (228 mg) three times a day; the entire course of treatment lasted three months.

[0038] Patients in the treatment group received a combination of mesenchymal stem cells and HP-16 peptides using a kit. Mesenchymal stem cells were administered intravenously at a dose of 1 million cells / kg body weight once a month, while HP-16 peptide was administered orally at a dose of 1 mg / kg body weight three times daily. The entire treatment course also lasted three months.

[0039] 2. Inclusion and Exclusion Criteria

[0040] Inclusion criteria: Meet the diagnostic criteria for NAFLD according to liver ultrasound examination; age between 18 and 65 years old; agree to participate in this study and sign the informed consent.

[0041] Exclusion criteria: patients with other liver diseases, such as viral hepatitis, autoimmune hepatitis, drug-induced liver injury, hereditary liver disease, etc.; patients with severe cardiovascular, kidney disease or other systemic diseases that may affect liver function and lipid metabolism; patients who have used drugs that may affect liver fat deposition in the past three months; pregnant or lactating women; patients who are allergic or intolerant to the study drugs; patients who have received treatment with the corresponding drugs of the present invention before the start of the study.

[0042] 3. Observation indicators

[0043] To evaluate the improvement in liver function and glucose and lipid metabolism in patients with nonalcoholic fatty liver disease (NAFLD) after comprehensive treatment, the following observational indicators were established. Patients were tested before and at the end of treatment. Liver function tests included alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). These indicators reflect hepatocyte damage and liver function. Liver function tests were performed using a fully automated biochemical analyzer. A 5 mL fasting morning venous blood sample was collected from all patients and centrifuged at 3500 rpm for 10 minutes. The supernatant was collected and assayed according to the assay kits and procedures provided by the biochemical analyzer manufacturer (Abbott, USA). Glucose and lipid metabolism indicators included fasting blood glucose (FBG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). Fasting blood glucose reflects the patient's glucose metabolism status, while lipid metabolism indicators indicate the patient's lipid storage and transport status. Glucose and lipid metabolism indicators were measured at the same time and using the same methods as liver function tests, using the corresponding fully automated biochemical analyzer and manufacturer-provided reagents. Throughout the study, patients were closely monitored, any adverse events were recorded, and their relationship to treatment was assessed, including drug-specific adverse effects and general discomfort reactions such as gastrointestinal discomfort, rash, and headache. After the treatment, all patients were evaluated for clinical efficacy, including marked effect, effectiveness, and ineffectiveness.

[0044] 4. Statistical significance

[0045] Data were reviewed for completeness and accuracy after collection. Continuous variables were described using mean ± standard deviation (mean ± SD), and comparisons between the two groups were performed using the independent sample t-test. Non-continuous variables were described using numbers and percentages, and comparisons between the two groups were performed using the chi-square test. Statistical analyses were performed using SPSS software. Throughout the study, all statistical analyses were two-sided with a significance level of 0.05.

[0046] 5. Experimental results

[0047] 5.1 Baseline Characteristics

[0048] Before treatment, there was no significant difference in age, gender distribution, and body mass index (BMI) between the two groups of patients (P>0.05), as shown in Table 1, indicating that the data of the two groups of patients were comparable.

[0049]

[0050] Table 1: Baseline characteristics and clinical data.

[0051] 5.2 Liver function indicators

[0052] Compared with the control group, the treatment group showed significant decreases in liver function indicators after treatment (P<0.05), including alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST). The ALP level in the treatment group decreased from 165.24±28.45 U / L before treatment to 118.67±22.13 U / L after treatment, while the control group decreased from 163.55±27.99 U / L to 146.88±25.62 U / L; the difference was significant (P<0.001). Moreover, the difference between the two groups was more obvious in ALT levels, with the ALT level in the treatment group decreasing from 52.84±14.76 U / L to 33. The AST levels in the treatment group decreased from 48.92±12.37U / L to 30.63±9.84U / L, while those in the control group decreased from 49.28±12.58U / L to 42.51±11.69U / L (P<0.001). Figure 1 .

[0053] 5.3 Glucose and lipid metabolism indicators

[0054] After treatment, the treatment group also showed significantly better improvements in glucose and lipid metabolism indicators than the control group (P < 0.05). Fasting blood glucose (FBG) in the treatment group decreased from 6.32 ± 1.25 mmol / L before treatment to 5.48 ± 0.89 mmol / L after treatment, while in the control group, it decreased from 6.28 ± 1.21 mmol / L to 6.01 ± 1.03 mmol / L. There was no significant difference between the two groups before treatment, but after treatment, the FBG in the treatment group was significantly lower than that in the control group (P = 0.001). Among lipid-related indicators, the total cholesterol (TC) level in the treatment group was significantly lower than that in the control group (treatment group: 4.65 ± 0.98 mmol / L, control group: 5.34 ± 1.02 mmol / L; P < 0.001). After treatment, the low-density lipoprotein cholesterol (LDL-C) in the treatment group also improved significantly compared to the control group (P<0.001), decreasing from 3.58±0.76mmol / L to 2.78±0.69mmol / L in the treatment group, while the control group saw a smaller decrease, from 3.62±0.79mmol / L to 3.40±0.73mmol / L. In addition, the improvement in high-density lipoprotein cholesterol (HDL-C) was also more significant in the treatment group (P<0.001), increasing from 1.25±0.28mmol / L to 1.48±0.30mmol / L, while in the control group, this indicator increased from 1.26±0.27mmol / L to 1.28±0.26mmol / L. Figure 2 .

[0055] 5.4 Clinical Efficacy Evaluation

[0056] In the treatment group, 35 patients (58.33%) achieved marked efficacy, while only 15 patients (25.00%) in the control group achieved marked efficacy (P < 0.001). The effective rate was 33.33% in the treatment group, compared with 50.00% in the control group (P < 0.001). The number of patients who experienced no effect was 8.33% in the treatment group, compared with 25.00% in the control group (P < 0.001). See Table 2.

[0057]

[0058] Table 2. Clinical efficacy evaluation

[0059] 5.5 Incidence of Adverse Reactions

[0060] During treatment, both groups reported some degree of adverse reactions. In the treatment group, the incidence of gastrointestinal discomfort was 8.33%, rash was 3.33%, and headache was 1.67%. In the control group, the incidence of gastrointestinal discomfort was 11.67%, rash was 5.00%, and headache was 1.67%. The overall incidence of adverse reactions in the two groups was 13.33% and 18.33%, respectively, and the differences did not reach statistical significance (P>0.05). See Table 3.

[0061]

[0062] Table 3: Comparison of adverse reaction rates

[0063] 6. Conclusion

[0064] The experimental results of the present invention showed that there was no significant difference in baseline characteristics between the two groups of patients before treatment, which provides a good premise for the objective evaluation of subsequent effects. After treatment, the treatment group with combined medication was superior to the control group in liver function indicators (ALP, ALT and AST). The levels of ALP, ALT and AST are often used as indicators to evaluate the degree of liver inflammation and damage. ALT and AST are enzymes in liver cells, and their elevated serum levels usually reflect liver cell damage. ALP is a marker of bile duct function, and its changes reflect cholestasis and liver excretion dysfunction. These indicators in the treatment group were significantly reduced after treatment, reflecting the additional benefits of mesenchymal stem cells and HP-16 polypeptide in liver protection. Mesenchymal stem cells combined with HP-16 polypeptide can maintain the integrity of liver cells, promote liver cell regeneration, reduce endotoxin damage to liver cells, regulate liver function and blood lipid levels, repair liver cell membranes, adjust metabolic abnormalities of fatty liver, and significantly improve liver disease symptoms; the combined use of the two can produce a synergistic effect, improving patients' fatty liver lesions from multiple aspects.

[0065] In terms of glucose and lipid metabolism indicators, the treatment group showed significantly greater improvements in FBG, TC, LDL-C, and HDL-C compared to the control group. Because NAFLD is closely associated with metabolic syndrome and its components, such as diabetes and hyperlipidemia, improvements in these indicators suggest that combined therapy is not only effective in reducing liver fat but also improves overall metabolic status and reduces the risk of cardiovascular disease. The decrease in fasting blood glucose suggests that combined therapy may have improved insulin sensitivity, while improvements in lipid indicators may reduce the risk of atherosclerosis, which is of great clinical significance.

[0066] Clinical efficacy evaluation further confirmed the effectiveness of this combined treatment approach, with the treatment group showing a significantly higher efficacy rate than the control group, while the inefficacy rate was significantly lower. This result further demonstrates that combined therapy with mesenchymal stem cells and HP-16 peptide is more effective than polyene phosphatidylcholine in improving the clinical symptoms and signs of NAFLD patients. Furthermore, in terms of adverse reactions, both groups experienced some degree of gastrointestinal discomfort, rash, and headache, but the difference in incidence was not statistically significant, indicating that mesenchymal stem cells and HP-16 peptide had no significant adverse effect on patient tolerance and that the combined therapy had an acceptable safety profile.

[0067] The embodiments described above are merely descriptions of preferred implementations of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the scope of protection determined by the claims of the present invention.

Claims

1. Use of mesenchymal stem cells and HP-16 polypeptide in the preparation of a drug kit for improving liver function in patients with non-alcoholic fatty liver disease, characterized in that: The amino acid sequence of the HP-16 polypeptide is: RTIGVYKSHLDPEECM; The mesenchymal stem cells are isolated and cultured by the following method: S1. Rinse fresh adipose tissue three times with PBS under sterile conditions to remove blood and impurities, and cut the adipose tissue into small pieces of 0.1 x 0.1 cm³. S2. Place the tissue fragment in a container containing 5 times the volume of collagenase type I and gently shake at 37°C to promote digestion for 40 minutes. Pipet with cell suspension every 10 minutes until the tissue is completely disintegrated. S3. After digestion is complete, an equal volume of DMEM high-glucose medium containing 10% fetal bovine serum was added to stop the digestion. The DMEM high-glucose medium was filtered through a 70-mesh filter and then centrifuged at 1200 rpm for 5 minutes. The supernatant was discarded and the cells were resuspended in PBS and washed three times to remove residual enzymes and impurities. S4. The cell suspension was cultured in a culture dish containing homemade MSC culture medium, which consisted of 1000 mL of DMEM high glucose, 2.5 μg / L bFGF, 200 mL of serum replacement, 10 mL of non-essential amino acids, and 10 mL of penicillin-streptomycin solution 100X, and cultured at 37°C and 5% CO2. S5. When the cells reach 80% confluence, digest them with 0.25% trypsin-EDTA solution for 2 min. Add an equal amount of DMEM high-glucose medium containing 10% fetal bovine serum to stop the digestion. Gently pipette to prepare a single-cell suspension. After centrifugation and washing, subculture the cells at a ratio of 1:3 to a new culture dish and continue culturing. S6. When the cells reach passage P3, mesenchymal stem cells are harvested.

2. The use according to claim 1, characterized in that: The HP-16 polypeptide is prepared by solid phase synthesis.

3. The use according to claim 1, characterized in that: The medicine kit also contains a pharmaceutically acceptable carrier.

4. The use according to claim 3, characterized in that: The carrier includes an excipient.

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