Application of hUC-MSCs combined with Met in preparation of anti-diabetic drugs

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of hUC-MSCs combined with Met in preparation of anti-diabetic drugs. The diabetes mellitus is particularly type 2 diabetes mellitus. Through construction of a T2DM rat model, paraffin tissue section detection, serum ELISA, cell experiments and the like, researches prove that T2DM can be effectively treated by HUC-MSCs combined with Met treatment, including reduction of body blood sugar and recovery of blood sugar regulation ability; liver injury caused by T2DM is relieved and repaired; the glycolipid metabolism of the liver of the T2DM rat is improved; the immune inflammation disorder caused by T2DM is relieved; and the apoptosis level of liver cells is improved. And compared with a single hUC-MSCs or Met treatment mode, the combined treatment mode has a better treatment effect. The invention provides a theoretical basis and a new treatment strategy for treatment of T2DM.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and specifically relates to an application of hUC-MSCs combined with Met in the preparation of an anti-diabetic drug. Background Art

[0002] Diabetes mellitus (DM) is a condition in which the body produces insufficient insulin or fails to respond normally to insulin, resulting in abnormally high blood sugar levels. Long-term uncontrolled blood sugar levels can lead to serious complications, including cardiovascular disease, stroke, kidney failure, blindness caused by retinopathy, nerve damage, foot ulcers, and even amputation. The disease is primarily categorized into three types: type 1, type 2, and gestational diabetes. Type 2 diabetes mellitus (T2DM) is the most common type, accounting for over 90% of cases.

[0003] Type 2 diabetes (T2DM), a globally prevalent, long-term, chronic metabolic disease, is difficult to fully cure. Its incidence has continued to rise in recent years, with a significant number of patients, many of whom face poor individual prognoses. Furthermore, T2DM is often accompanied by complications such as impaired liver and pancreatic function, which severely impact patients' quality of life. Currently, the treatment of T2DM relies primarily on a combination of oral hypoglycemic medications and lifestyle and nutritional adjustments. Metformin (N,N-dimethylbiguanide) is the most widely used oral hypoglycemic agent and is recommended as first-line treatment for all newly diagnosed patients with T2DM. This drug, a biguanide antidiabetic agent, was originally derived from galegine (isopentenylguanidine), a guanidine derivative discovered in the French clove Galegao fficinalis. Since its initial launch in France in 1957, it has held a pivotal position in diabetes treatment. Its mechanism of action is primarily through improving insulin sensitivity. By enhancing insulin signaling, it promotes glucose uptake and utilization in peripheral tissues such as skeletal muscle and adipose tissue, while inhibiting hepatic glucose output, thereby effectively lowering blood glucose levels. However, although metformin can control blood sugar, it cannot repair damaged pancreatic function, nor can it prevent lesions in tissues such as the liver and pancreas, or restore the functions of related organs.

[0004] With the advancement of stem cell and regenerative medicine, human umbilical cord mesenchymal stem cells (hUC-MSCs) have become an ideal cell type for diabetes treatment due to their abundant origin, primordial properties, immunomodulatory, non-immunogenicity, secretory and paracrine functions, migration, proliferation, and multipotency. Human umbilical cord (HUC) is considered a high-quality source for the isolation, culture, and expansion of MSCs. MSCs isolated from HUC are readily available, ethically sound, and available in a virtually unlimited supply, whereas they are often discarded. HUC-derived MSCs are low-cost and non-invasive to obtain, have a high proliferation rate, can be cryopreserved for long periods, exhibit a low immunogenic phenotype and possess immunosuppressive properties, and can be harvested from a growth factor-rich matrix. Furthermore, hUC-MSCs express multiple stem cell genes and can differentiate into various mature cell types, including neurons, cells with bone and osteoblastic phenotypes, cardiomyocytes, chondrocytes, and adipocytes, reinforcing their multipotency. Like bone marrow MSCs, hUC-MSCs can also give rise to adult cells of ectodermal and mesodermal origin. In conclusion, hUC-MSCs have great potential in the treatment of diabetes.

[0005] Currently, there are relatively few studies on the combined use of hUC-MSCs and Met in the treatment of T2DM. Developing a combination therapy that can effectively treat T2DM and its complications is of great value. Summary of the Invention

[0006] In light of this, one of the objectives of the present invention is to provide a method for the use of human umbilical cord mesenchymal stem cells (hUC-MSCs) in combination with metformin in the preparation of a medicament for the treatment of diabetes. The present invention has found that the combination of hUC-MSCs and Met for the treatment of diabetes is more effective than treatment with hUC-MSCs or Met alone.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a drug for treating diabetes.

[0009] Preferably, the diabetes is type 2 diabetes.

[0010] Preferably, the human umbilical cord mesenchymal stem cells highly express CD90, CD73 and CD105, and lowly express CD34, CD45 and HLA-DR.

[0011] More preferably, the expression rates of CD90, CD73 and CD105 in the human umbilical cord mesenchymal stem cells are all ≥99%, and the expression rates of CD34, CD45 and HLA-DR are all ≤1%.

[0012] Preferably, the method for preparing human umbilical cord mesenchymal stem cells comprises: obtaining neonatal umbilical cord tissue, digesting it with collagenase to obtain a cell precipitate; adding cell culture medium to the cell precipitate for culturing to obtain human umbilical cord mesenchymal stem cells.

[0013] Preferably, the cell culture medium is MEM medium.

[0014] As a preferred solution, the human umbilical cord mesenchymal stem cells are prepared by the following method:

[0015] (1) Umbilical cord tissue processing: Take the umbilical cord tissue of the newborn, wash the blood on the surface of the umbilical cord tissue with physiological saline, cut it into 3-5 cm small segments; separate and remove the blood vessels, keep the Wharton's jelly tissue and cut it into 1 mm 3 Add appropriate amount of collagenase to digest for 1-2 hours until the tissue blocks are basically discrete. Filter the digested cell suspension through a filter to remove undigested tissue residues and centrifuge to obtain the cell pellet.

[0016] (2) Cell culture: MEM medium was added to the cell pellet, and the cell pellet was resuspended and inoculated into a culture flask, which was then cultured in a CO2 incubator to obtain human umbilical cord mesenchymal stem cells;

[0017] (3) Dilute human umbilical cord mesenchymal stem cells (10 mM) with cell culture medium at a ratio of 1:500 to obtain 2× EdU working solution; add an equal volume of 2× EdU working solution preheated at 37°C to a 6-well plate and continue incubating the cells overnight;

[0018] (4) Preparation of hUC-MSCs injection suspension: Select cells labeled with EdU overnight, digest them with trypsin, and add culture medium to terminate the digestion; dilute the cells to 1×10 6 After thorough mixing, hUC-MSCs injection suspension was prepared.

[0019] A second object of the present invention is to provide a use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a drug for lowering blood sugar and / or restoring blood sugar regulation ability.

[0020] The third object of the present invention is to provide a use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a drug for treating liver damage, liver lipid metabolism disorders, liver glucose metabolism disorders and / or immune inflammatory disorders caused by type 2 diabetes.

[0021] To achieve the above object, the present invention adopts the following technical solutions:

[0022] Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a medicament for treating liver damage, liver lipid metabolism disorder, liver glucose metabolism disorder and / or immune inflammatory disorder caused by type 2 diabetes.

[0023] Preferably, the drugs include: drugs that reduce abnormally elevated AST and ALT levels in serum, drugs that increase HDL-C levels in the liver, drugs that reduce LDL-C levels in the liver, drugs that reduce IL-6 and TNF-α levels in serum, drugs that increase IL-10 levels in serum, drugs that reduce excessive glycogen deposition in the liver, drugs that reduce ICAM-1 protein expression in the liver and / or drugs that increase CPT1A protein expression in the liver.

[0024] A fourth object of the present invention is to provide a use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a drug for inhibiting abnormal apoptosis of liver cells caused by type 2 diabetes.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a drug for inhibiting abnormal apoptosis of liver cells caused by type 2 diabetes.

[0027] Preferably, the drugs include: drugs that increase the expression of Bcl-2 protein in the liver, and drugs that decrease the expression of Bax and caspase3 proteins in the liver.

[0028] A fifth object of the present invention is to provide a pharmaceutical composition for treating type 2 diabetes and its complications.

[0029] To achieve the above object, the present invention adopts the following technical solutions:

[0030] A pharmaceutical composition for treating type 2 diabetes and its complications, the composition comprising metformin and human umbilical cord mesenchymal stem cells; the complications include any one or more of hyperglycemia, diabetic liver damage, liver glucose and lipid metabolism disorders, and immune inflammatory disorders.

[0031] The beneficial effects of the present invention are:

[0032] This study focused on the treatment of a rat model of T2DM using hUC-MSCs combined with Met. Detection of liver histological changes, related protein expression, and changes in serum metabolic markers confirmed that hUC-MSCs combined with Met were more effective in treating T2DM than either hUC-MSCs or Met alone. Specific findings:

[0033] (1) hUC-MSCs+Met combined treatment can effectively lower blood sugar and restore blood sugar regulation ability.

[0034] (2) hUC-MSCs combined with Met can effectively improve the body's liver metabolism level, significantly enhance the liver's ability to regulate and repair lipid metabolism, and have a good alleviating and repairing effect on liver lipid metabolism disorders and related liver damage caused by T2DM, providing a highly potential intervention strategy for the treatment of abnormal lipid metabolism and liver lesions in T2DM patients.

[0035] (3) hUC-MSCs combined with Met therapy can effectively regulate the body's immune inflammation level and has a good alleviating and repairing effect on the immune inflammatory disorder caused by T2DM. Data show that combined therapy can effectively reduce serum AST and ALT levels, and normalize HDL-C and LDL-C levels, while regulating the levels of inflammatory factors such as IL-6, TNF-α, and IL-10.

[0036] (4) hUC-MSCs combined with Met treatment can effectively improve the glucose metabolism in the liver of T2DM rats, significantly reduce excessive glycogen deposition, and is more conducive to restoring the normal function of liver tissue in glycogen metabolism.

[0037] (5) hUC-MSCs combined with Met treatment improved liver function in T2DM rats through the MAPK signaling pathway. Specifically, the combined treatment regulated the expression of key proteins in the MAPK signaling pathway and glucose and lipid metabolism. Among them, the expression of P-p38MAPK, P-ERK1 / 2, PPARA, AMPKα, and HK2 proteins was upregulated, while the expression of JNK and SREBF1 proteins was downregulated.

[0038] (6) hUC-MSCs+Met combined treatment can effectively improve the apoptosis level of liver cells. Western blot analysis showed that after combined treatment, the expression of Bcl-2 protein in liver tissue increased, while the expression of Bax and caspase3 proteins decreased; TUNEL staining results showed that the number of TUNEL-positive cells decreased.

[0039] In summary, this invention not only fills a gap in animal experimental research in this field, providing key data support for subsequent preclinical studies, but also provides clues for further exploration of the mechanism of action of combination therapy and lays a theoretical foundation for the development of more effective T2DM treatment strategies. This invention has important scientific significance and clinical application value in promoting innovation in T2DM treatment methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 EdU staining images of liver paraffin tissue sections of rats at D3, D7, and D14 (scale bar = 50 μm);

[0041] Figure 2EdU staining images of pancreatic paraffin tissue sections from rats at D3, D7, and D14 (scale bar = 50 μm);

[0042] Figure 3 The morphology of hUC-MSCs at passage 4 under light microscopy (scale bar = 100 μm);

[0043] Figure 4 Figure 2 is the result of hUC-MSCs osteogenic, adipogenic and chondrogenic differentiation identification (scale bar = 50 μm); Figure 4 -A is an Alizarin red staining image of undifferentiated hUC-MSCs cells; Figure 4 -B is an Alizarin red staining image of hUC-MSCs cells cultured for 18 days after osteogenic differentiation;

[0044] Figure 4 -C is the Oil Red O staining image of undifferentiated hUC-MSCs cells; Figure 4 -D is the Oil Red O staining of hUC-MSCs cells cultured for 20 days after adipogenic differentiation; Figure 4 -E is the Alcian blue staining image of undifferentiated hUC-MSCs cells; Figure 4 -F is the Alcian blue staining of hUC-MSCs cells cultured for 20 days after chondrogenic differentiation;

[0045] Figure 5 Figure 2 is a graph showing the expression of hUC-MSCs surface markers detected by flow cytometry; Figure 5 -A is a graph showing the expression of hUC-MSCs surface marker CD90 detected by flow cytometry; Figure 5 -B is a graph showing the expression of hUC-MSCs surface marker CD73 detected by flow cytometry; Figure 5 -C is a graph showing the expression of hUC-MSCs surface marker CD105 detected by flow cytometry; Figure 5 -D is a graph showing the expression of hUC-MSCs surface marker CD34 detected by flow cytometry; Figure 5 -E is a graph showing the expression of hUC-MSCs surface marker CD45 detected by flow cytometry; Figure 5 -F is a graph showing the expression of hUC-MSCs surface marker HLA-DR detected by flow cytometry;

[0046] Figure 6 The figure shows the test results of the effects of high-sugar and high-fat diet combined with STZ on the physiological indicators of SD rats; Figure 6 -A is the test result of body weight of SD rats in NCD group and HFD group; Figure 6 -B is the test results of blood glucose changes in rats in the NCD group and HFD+STZ group; Figure 6-C is the result of IPGTT experiment; Figure 6 -D is the IPITT experimental result diagram;

[0047] Figure 7 This is a diagram showing the enrichment of signaling pathways in the liver of type 2 diabetic rats using KEGG enrichment analysis;

[0048] Figure 8 This is the result diagram for detecting the effects of hUC-MSCs and hUC-MSCs+Met treatment on the physiological indicators of T2DM rats; Figure 8 -A is the test result of the body weight of rats in each group after injection of hUC-MSCs; Figure 8 -B is the blood glucose test results of rats in each group after injection of hUC-MSCs; Figure 8 -C is the IPGTT test results of rats in each group after injection of hUC-MSCs; Figure 8 -D is the IPITT test results of rats in each group after injection of hUC-MSCs;

[0049] Figure 9 This is the test result diagram of serum biochemical index-liver function; Figure 9 -A is the AST level test result diagram; Figure 9 -B is the ALT level test result diagram;

[0050] Figure 10 This is the test result diagram of serum biochemical index-blood lipid level; Figure 10 -A is the result graph of HDL-C level test; Figure 10 -B is the result of LDL-C level test;

[0051] Figure 11 The results of ELISA kits for determining the levels of inflammatory factors in serum (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001); Figure 11 -A is the result of IL-10 level detection; Figure 11 -B is the result of IL-6 level detection; Figure 11 -C is the result of TNF-α level detection;

[0052] Figure 12 The results of HE staining to detect the histopathological changes in rats treated with hUC-MSCs and hUC-MSCs+Met (scale bar = 50 μm); Figure 12 -A is the HE staining image of rats in the NC group; Figure 12 -B is the HE staining image of rats in the T2DM group; Figure 12 -C is the HE staining image of rats in the Met group; Figure 12-D is the HE staining image of rats in the MSC group; Figure 12 -E is the HE staining image of rats in the MSC+Met group;

[0053] Figure 13 The results of PAS staining for detecting glycogen deposition in rat liver tissue treated with hUC-MSCs and hUC-MSCs+Met (scale bar = 50 μm); Figure 13 -A is the PAS staining image of liver tissue of rats in the NC group; Figure 13 -B is the PAS staining image of liver tissue of rats in the T2DM group; Figure 13 -C is the PAS staining image of liver tissue of rats in the Met group; Figure 13 -D is the PAS staining image of liver tissue of rats in the MSC group; Figure 13 -E is the PAS staining image of liver tissue of rats in the MSC+Met group; Figure 13 -F is the detection result of PAS staining positive area of ​​liver glycogen staining of rats in each group;

[0054] Figure 14 The results of IHC detection of the distribution of ICAM-1 protein expression in T2DM rat liver tissue and the IDO value of ICAM-1 protein expression; Figure 14 -A is the result of IHC detection of the distribution of ICAM-1 protein expression in the liver tissue of rats in the NC group;

[0055] Figure 14 -B is the result of IHC detection of the distribution of ICAM-1 protein expression in the liver tissue of rats in the T2DM group; Figure 14 -C is the result of IHC detection of the distribution of ICAM-1 protein expression in the liver tissue of rats in the Met group; Figure 14 -D is the result of IHC detection of the distribution of ICAM-1 protein expression in the liver tissue of rats in the MSC group; Figure 14 -E is the result of IHC detection of the distribution of ICAM-1 protein expression in the liver tissue of rats in the MSC+Met group; Figure 14 -F is the result graph of IDO value of ICAM-1 protein expression in liver tissue of rats in each group;

[0056] Figure 15 The results of IHC detection of the distribution of CP1T1A protein expression in T2DM rat liver tissue and the IDO value of CP1T1A protein expression; Figure 15 -A is the result of IHC detection of the distribution of CP1T1A protein expression in the liver tissue of rats in the NC group;

[0057] Figure 15-B is the result of IHC detection of the distribution of CP1T1A protein expression in the liver tissue of rats in the T2DM group; Figure 15 -C is the result of IHC detection of the distribution of CP1T1A protein expression in the liver tissue of rats in the Met group; Figure 15 -D is the result of IHC detection of the distribution of CP1T1A protein expression in the liver tissue of rats in the MSC group; Figure 15 -E is the result of IHC detection of the distribution of CP1T1A protein expression in the liver tissue of rats in the MSC+Met group; Figure 15 -F is the result graph of IDO value of CP1T1A protein expression in liver tissue of rats in each group;

[0058] Figure 16 This is the experimental test result of hUC-MSCs and hUC-MSCs+Met treatment improving the liver function of T2DM rats through MAPK signaling pathway; Figure 16 -A and Figure 16 -B is the result of Western Blot experiment detecting the changes of MAPK signaling pathway and liver lipid metabolism related proteins after treatment; Figure 16 -C is the morphological diagram of the liver in each group; Figure 16 -D is the result of Image J analysis of Western Blot experiment to detect the relative expression of p38 MAPK / β-actin after treatment; Figure 16 -E is the result of Image J analysis of Western Blot experiment to detect the relative expression of P-p38 MAPK / β-actin after treatment; Figure 16 -F is the result of Image J analysis of Western Blot experiment to detect the relative expression of P-ERK1 / 2 / β-actin after treatment; Figure 16 -G is the result of Image J analysis of Western Blot experiment to detect the relative expression of JNK / β-actin after treatment; Figure 16 -H is the result of Image J analysis of Western Blot experiment to detect the relative expression of PPARA / β-actin after treatment; Figure 16 -I is the result of Image J analysis of Western Blot experiment to detect the relative expression of AMPKα / β-actin after treatment; Figure 16 -J is the result of Image J analysis of Western Blot experiment to detect the relative expression of HK2 / β-actin after treatment; Figure 16 -K is the result of Image J analysis of Western Blot experiment to detect the relative expression of SREBF1 / β-actin after treatment;

[0059] Figure 17 The figure shows the results of Western Blot assay to detect the changes and relative expression levels of proteins related to apoptosis pathway in hUC-MSCs and combined treatment; Figure 17 -A is the result of Western Blot assay to detect changes in proteins related to apoptosis pathway after treatment; Figure 17 -B is a graph showing the relative expression of Bax / β-actin detected by Western Blot assay; Figure 17 -C is a graph showing the relative expression of Bcl-2 / β-actin detected by Western Blot assay; Figure 17 -D is a graph showing the relative expression of caspase3 / β-actin detected by Western Blot assay;

[0060] Figure 18 The figure shows the results of TUNEL staining to detect cell apoptosis in rat liver tissue after hUC-MSCs and hUC-MSCs+Met treatment (scale bar = 50 μm);

[0061] In the above figures, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. DETAILED DESCRIPTION

[0062] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0063] In the embodiments of the present invention, unless otherwise specified, the instruments, reagents, drugs, etc. involved can be purchased through conventional commercial channels.

[0064] This study focused on the treatment of T2DM rat models with hUC-MSCs and hUC-MSCs combined with Met. The efficacy of the combined treatment was assessed by examining liver histological changes, related protein expression, and changes in serum metabolic markers. The main research topics in this example are as follows:

[0065] (1) Construction of a type 2 diabetes rat model: A T2DM rat model was constructed by feeding the rats with a high-sugar and high-fat diet combined with intraperitoneal injection of a small dose of streptozotocin (STZ). During the modeling process, the rats' body weight, blood sugar and other indicators were regularly monitored to ensure the successful construction of the model. At the same time, a normal control group was established and fed with ordinary feed. After the model stabilized, comprehensive physiological index tests were performed on the two groups of rats, including intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPTTT), to confirm the effectiveness and stability of the model and provide a reliable animal model basis for subsequent studies.

[0066] (2) Screening of differentially expressed genes and enriched signal pathways: Through the GEO database and other bioinformatics resources, the gene expression data of normal rats and T2DM rats were deeply analyzed to screen out differentially expressed genes and conduct signal pathway enrichment analysis on the differentially expressed genes, preliminarily exploring the potential molecular mechanisms related to the pathogenesis of T2DM and providing a theoretical basis for subsequent research.

[0067] (3) Evaluation of the effect of treatment on the improvement of liver lipid metabolism: T2DM model rats were divided into T2DM group, Met group, MSC group, MSC+Met group, and NCD group (i.e., NC group, Normal Control) rats into 5 groups, 6 rats in each group (n=6), and received corresponding treatment. After the treatment, the liver tissues of the rats were obtained. Periodic acid Schiff (PAS) staining was used to visually display the uptake and use of glycogen in the liver; hematoxylin-eosin (HE) staining was performed to observe the morphological changes of liver tissue and comprehensively evaluate the improvement effect of treatment on liver tissue damage; protein immunoblotting (WB) technology was used to detect the expression level changes of key proteins of the MAPK signaling pathway, key proteins related to lipid metabolism, and representative proteins of apoptosis in the liver; immunohistochemistry (IHC) experiments were used to observe the localization and expression differences of lipid metabolism-related proteins in liver tissue.

[0068] (4) Evaluation of the improvement effect of treatment on serum-related indicators: The serum of rats in each group was collected, and the enzyme-linked immunosorbent assay (ELISA) technique was used to detect changes in liver function indicators, lipid density protein levels, and inflammatory factor levels, and the effect of treatment on the overall health status and metabolic level of T2DM rats was evaluated from a serological perspective.

[0069] (5) Verification of therapeutic effects and exploration of therapeutic mechanisms: Based on the above serum and liver test results, the therapeutic effects of hUC-MSCs, Met, and their combination on T2DM rats were verified from multiple aspects and levels. Combined with the KEGG enrichment signaling pathway, the changes in the expression of proteins related to the MAPK signaling pathway were used to explore the mechanism of action of the combined intervention on the molecular and tissue levels, providing a theoretical basis and new treatment strategy for the clinical treatment of T2DM with hUC-MSCs combined with Met.

[0070] In the embodiments of the present invention, the main research results are as follows:

[0071] This study conducted a series of experiments focused on establishing a T2DM rat model, evaluating the therapeutic effects of hUC-MSCs and hUC-MSCs + Met, and exploring related mechanisms. A high-sugar, high-fat diet combined with intraperitoneal injection of a low-dose streptozotocin (STZ) was used to establish a T2DM rat model. Physiological indicators such as body weight and blood glucose were monitored, along with glucose tolerance and insulin tolerance, to verify the success of the model. This model provides a reliable experimental foundation for subsequent research, enabling more targeted and scientific studies of the pathogenesis and treatment of T2DM.

[0072] This experiment focused on studying the improvement effect of combined therapy on the liver of T2DM rats. General physiological indicators showed that after treatment with hUC-MSCs and hUC-MSCs+Met, the rats regained weight, reduced blood sugar, and significantly restored their blood sugar regulation ability, among which the MSC+Met group had the best effect. This shows that combined therapy can effectively improve the metabolic disorder of T2DM rats and promote the body's recovery to health. ELISA results showed that combined therapy significantly reduced the levels of AST and ALT in serum, and the levels of HDL-C and LDL-C returned to normal, while regulating the levels of inflammatory factors such as IL-6, TNF-α and IL-10. This series of changes shows that combined therapy has improved liver function, effectively regulated lipid metabolism, reduced the levels of pro-inflammatory factors and increased the levels of anti-inflammatory factors, and effectively alleviated the damage to the liver caused by T2DM.

[0073] Pathological features were identified by staining of liver tissue sections. HE staining showed significant improvement in liver tissue damage, with normal cell morphology and arrangement. PAS staining indicated decreased glycogen deposition, alleviated glucose metabolism disorders, and normalized glycogen uptake and utilization efficiency. Immunohistochemistry confirmed normalized expression of the lipid metabolism-related proteins ICAM-1 and CPT1A, and a reduced inflammatory response. ICAM-1 binds to integrins on the leukocyte surface, promoting leukocyte recruitment and infiltration into the liver, triggering hepatic inflammation. This inflammatory microenvironment interferes with the insulin signaling pathway, inhibiting tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), and thereby impairing insulin-stimulated glucose uptake and glycogen synthesis. After combined treatment, ICAM-1 levels decreased and insulin levels increased, suggesting that decreased ICAM-1 no longer inhibits insulin production. CPT1A, as the rate-limiting enzyme in fatty acid β-oxidation, altered activity directly affects hepatic fatty acid metabolism and energy supply, thereby affecting glucose metabolism processes such as gluconeogenesis. After combined treatment, it was shown that the distribution of CPT1A in liver cells increased, indicating that the efficiency of fatty acid β-oxidation increased, fat accumulation in the liver decreased, insulin resistance was reduced, and the gluconeogenesis process returned to normal, so that blood sugar was effectively controlled, thereby restoring the balance of glucose and lipid metabolism.

[0074] Western blot analysis verified the molecular mechanisms involved, and the results showed that combined treatment modulated the expression of key proteins in the MAPK signaling pathway and lipid metabolism. Specifically, the expression of P-p38MAPK, P-ERK1 / 2, PPARA, AMPKα, and HK2 proteins was upregulated, while the expression of JNK and SREBF1 proteins was downregulated. P-p38MAPK, the phosphorylated activated form of P38MAPK, plays a crucial role in cellular stress and inflammatory signaling. Its upregulation suggests enhanced cellular resistance and a balanced inflammatory response, without disrupting lipid metabolism. P-ERK1 / 2 participates in signal transduction for various physiological processes, including cell proliferation and differentiation. Its increased expression promotes liver cell repair and regeneration, restoring liver function. PPARA plays a key role in regulating fatty acid metabolism and energy balance. Its increased expression promotes fatty acid metabolism and helps reduce hepatic fat accumulation. AMPKα, a key sensor and regulator of cellular energy status, promotes glucose uptake and utilization, enhances insulin sensitivity, and improves glucose metabolism. HK2, a key enzyme in glycolysis, participates in the phosphorylation of glucose. Increased HK2 expression enhances glycolysis and further improves glucose metabolism. JNK plays a crucial role in regulating cellular stress, apoptosis, and inflammatory signaling pathways. Decreased HK2 expression helps alleviate cellular stress and inflammation, protecting liver cells. SREBF1 is primarily involved in regulating lipogenesis. Decreased HK2 expression reduces lipogenesis and helps improve hepatic lipid metabolism.

[0075] Combined treatment improved the level of apoptosis in liver cells. Western blot analysis revealed that after treatment, the expression of Bcl-2 protein in liver tissue increased, while the expression of Bax and caspase3 proteins decreased; TUNEL staining results showed a decrease in the number of TUNEL-positive cells. Bcl-2 is an anti-apoptotic protein that can inhibit the release of cytochrome C from mitochondria, preventing the occurrence of apoptosis; Bax is a pro-apoptotic protein that can form a heterodimer with Bcl-2. When Bax expression increases, it disrupts the balance between pro-apoptotic and anti-apoptotic proteins in the cell, prompting the cell to undergo apoptosis; caspase3 is a key execution protease in the apoptosis process; its increased activity and expression accelerate the process of apoptosis. By regulating the expression of these apoptosis-related proteins, combined treatment reshapes the apoptosis regulatory network in liver cells, inhibits excessive apoptosis of liver cells, and helps the liver restore normal physiological function.

[0076] In summary, this study revealed the therapeutic effect and potential mechanism of hUC-MSCs combined with Met in the treatment of T2DM rats through multi-faceted experiments, providing a more solid theoretical basis and effective treatment strategy for the clinical treatment of T2DM.

[0077] Example 1. Culture, identification and directed differentiation of hUC-MSCs

[0078] (1) Experimental methods

[0079] (1) Acquisition of clinical samples

[0080] The human umbilical cord mesenchymal stem cells used in this study were obtained from a clear and compliant source. hUC-MSCs were collected from umbilical cord tissue of healthy mothers at Chongqing No. 9 People's Hospital. The entire stem cell acquisition process strictly adhered to relevant ethical and regulatory requirements. All procedures were complete, and relevant documents are included in the attached appendix for review and approval. The ethics review number for the collection and use of stem cells by Chongqing No. 9 People's Hospital for this study is: 2024-KeW(Lun Shen)-001.

[0081] (2) Acquisition and extraction of hUC-MSCs

[0082] The Department of Obstetrics and Gynecology at Chongqing Ninth People's Hospital is responsible for collecting umbilical cord tissue. They screen eligible umbilical cord donors and collect the tissue within 10 minutes of their infant's natural birth or cesarean section, following routine ligation and cutting of the umbilical cord to ensure its vitality and quality. After collection, medical staff disinfect and clean the tissue, place it in an umbilical cord collection bottle, tighten the cap, seal it, and seal it in a biosafety bag. The bag is then placed in an umbilical cord collection kit and stored at a constant temperature between 2°C and 10°C. Freezing is strictly prohibited to prevent radiation, collision, or contamination. The umbilical cord tissue must be delivered within 24 hours.

[0083] Umbilical cord tissue processing: Wash the blood on the surface of the umbilical cord tissue with physiological saline and cut it into 3-5cm small segments. Separate and remove the blood vessels in the clean bench, retain the Wharton's jelly tissue and cut it into 1mm 3 Add appropriate amount of collagenase to small pieces of tissue and digest for 1-2 hours until the tissue pieces are basically discrete. Filter the digested cell suspension through a filter to remove undigested tissue residues and centrifuge to obtain the cell pellet.

[0084] Cell Culture: Add MEM medium to the cell pellet, resuspend, and inoculate into a culture flask for culture in a CO2 incubator. Cell Cryopreservation: Collect and resuspend mesenchymal stem cells in the logarithmic growth phase, mix with freezing solution at a 1:1 ratio, and dispense into cryovials. Label with the relevant information and follow a cooling schedule of 4°C for 30 minutes, -20°C for 2 hours, and then transfer to -80°C overnight. Finally, place in a liquid nitrogen tank for long-term storage. Those unsuitable for cryopreservation should be discarded according to medical procedures.

[0085] Culture an appropriate number of cells in a 6-well plate. After the cells have been cultured overnight and have returned to normal, perform the desired drug treatment or other stimulation.

[0086] Dilute EdU (10 mM) 1:500 with cell culture medium to create a 2× EdU working solution. Add an equal volume of 2× EdU working solution preheated at 37°C to a 6-well plate. Continue incubating the cells overnight.

[0087] Preparation of hUC-MSCs injection suspension: Select cells labeled with EdU overnight, digest with trypsin, add culture medium to terminate digestion, and count the cells using a cell counting plate. Dilute the cells to 1×10 with physiological saline according to experimental requirements. 6 After thorough mixing, an injection suspension was prepared and used in the successfully established T2DM rat model.

[0088] (3) hUC-MSCs cell markers detected by flow cytometry

[0089] Cell morphology identification showed that the cells grew in a long spindle shape and adhered to the wall. After proliferation and passage, a large number of cells were fibroblast-like. The cells grew densely arranged in a vortex shape with obvious directionality, and had typical morphological characteristics of mesenchymal stem cells.

[0090] Detect the expression of cell surface markers, including positive surface markers CD90 and CD73 marked as APC, positive surface marker CD105 marked as PE; negative surface marker CD34 marked as PerCP, negative surface marker CD45 and HLA-DR marked as PE; CD90-APC and CD73-APC correspond to APC-labeled control antibody IgG1; CD105-PE, CD45-PE, HLA-DR-PE correspond to PE-labeled control antibody IgG1; CD34-PerCP corresponds to PerCP-labeled control antibody IgG1; and a blank control group was set up at the same time. The specific process of flow cytometry detection is as follows:

[0091] 1) Digest the cells with 0.25% trypsin for 3 minutes, centrifuge at 1500 rpm for 5 minutes, discard the supernatant, and resuspend the cells in sterilized PBS to prepare a cell suspension.

[0092] 2) After resuspending the cells, take 100 μL and add it to the labeled EP tube and mix well.

[0093] 3) Check the antibody sequence, add 5 μL for labeling, and incubate in the dark for 30 minutes.

[0094] 4) After incubation, add 500 μL PBS to each tube, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and repeat twice.

[0095] 5) Add 300 μL PBS to each tube, mix well, and test on the instrument.

[0096] (4) Directed differentiation of hUC-MSCs

[0097] hUC-MSCs possess strong multidirectional differentiation potential and can be induced to differentiate into a variety of different cell types. Differentiation can be directly induced by administering specific growth factors, culture medium components, or other stimulants to promote the development of stem cells toward specific cell types. Appropriate induction conditions and culture protocols are typically selected based on research or clinical needs to achieve differentiation into specific cell types, such as osteoblasts or adipocytes. Experimental methods are generally recommended in the literature.

[0098] according to The osteogenic differentiation kit requires that Osteogenic Induction Supplement A be thawed at room temperature, then added to the basal culture medium in a clean hood and mixed thoroughly. Rinse the tube and shake again. Seal, wrap, and label the tube. Before use, briefly centrifuge Osteogenic Induction Supplement B and add the mixture in the appropriate proportions to prepare complete osteogenic induction medium.

[0099] hUC-MSCs of passage 4 were taken and 1×10 4Cells were seeded into 12-well plates at a concentration of 100 cells / well. After adding maintenance medium, the cells were cultured in an incubator. When the cell confluence reached 80%-90%, complete osteogenic induction medium was used and the medium was changed every 3 days.

[0100] After about 20 days of induction, the culture medium was removed, the cells were rinsed with PBS, fixed with 4% paraformaldehyde, and rinsed again with PBS. Alizarin red staining was performed, and after rinsing with distilled water, calcium nodules were observed under a microscope to determine the effect of osteogenic differentiation.

[0101] according to The osteogenic differentiation kit requires the preparation of culture medium and induction differentiation medium A: Add serum, additives AI, and additives A-II to the basal culture medium in a clean bench. Wash the container with a small amount of basal culture medium and pour it in. After shaking, seal, wrap, and label the container.

[0102] Treat a six-well plate with 0.1% gelatin solution, seed the cells to be induced, and culture in standard complete medium. Once the cells are confluent, aspirate the medium and induce with Solution A for 3 days, followed by Solution B for 1 day. Alternate between Solution A and Solution B, observing the cells daily and adjusting the ratio of Solution A to B as needed until sufficient lipid droplets of appropriate size are present.

[0103] After induction, discard the culture medium and gently wash the cells 2-3 times with 1× PBS. Fix with 4% paraformaldehyde at room temperature for 30 minutes, then gently wash 2-3 times with 1× PBS. Prepare the working solution from the Oil Red O stock solution according to the proportion, centrifuge and remove the supernatant, add 2mL of the working solution to each well and stain at room temperature for 30 minutes. Discard the Oil Red O staining solution, gently wash 2-3 times with 1× PBS, add 2mL of 1× PBS, and observe the adipogenesis effect under a microscope. If storage is required, seal with sealing film and store at 4°C. The storage time should not exceed 1 week.

[0104] according to After preparing the premix of the chondrogenic differentiation kit and the complete medium, induce 3-4×10 5 Each cell to be induced is centrifuged, washed, and resuspended in complete culture medium. Place the cells in a sterile 15ml centrifuge tube and culture in an incubator without shaking. Once the cells have aggregated, gently flick them to suspend the chondrocytes. Change the culture medium every 2-3 days. Once chondrocytes with a diameter of 1.5-2 mm have been induced, they are fixed, dehydrated, transparentized, waxed, embedded, sectioned, mounted, dewaxed, and stained. Finally, observe the results of the alicein blue staining under a microscope.

[0105] (2) Experimental results

[0106] (1) Comparison of EdU fluorescence staining of the liver and pancreas of the two groups of rats at different time points

[0107] EdU staining is a new detection method. Its principle is that during the cell proliferation process, along with the semi-conservative replication of DNA, EdU (5-ethynyl-2'deoxyuridine) is incorporated into the newly synthesized DNA chain. Subsequently, EdU reacts with fluorescent azide by means of a covalent binding mechanism, thereby achieving fluorescent labeling of S phase cells, thereby reflecting the proliferation and survival status of the cells. In EdU staining, positive cells show the characteristic of strong green fluorescence in the cell nucleus. This experiment uses Hoechst, a specific stain for the cell nucleus. EdU staining is highly specific and only stains the cell nucleus without staining the cytoplasm. EdU and Hoechst are co-localized in the cell nucleus. The brightness and number of positive localization in EdU-stained tissue sections can intuitively reflect the proliferation of cells. The stronger the cell vitality and the greater the number, the higher the brightness and number of positive localization.

[0108] In this experiment, human umbilical cord mesenchymal stem cells (hUC-MSCs) were selected and infused into SD rats via the tail vein. EdU staining was performed on the liver and pancreas paraffin tissue sections of the rats on the third, seventh, and fourteenth days after infusion. Figure 1 and Figure 2 As shown in the figure, EdU-positive cells were observed at all time points. Specifically, on the third day, the number of EdU-positive cells was high and the fluorescence brightness was high. On the seventh day, the number of positive cells decreased and the fluorescence brightness also weakened. By the fourteenth day, EdU-positive cells had almost disappeared, the number was very small, and the fluorescence was weak.

[0109] (2) Identification of hUC-MSCs

[0110] The cultured cells were passaged at a ratio of 1:1. After 12 hours, most of the cells completed adhesion, but these adhered cells did not stretch outward, but took on a triangular or diamond shape. As the culture time advanced to 48 hours, the adhered cells quickly entered the proliferation stage, and their morphology gradually became consistent, turning into a long spindle shape. When the culture time reached 72 hours, the cell fusion rate reached 80%-90%, and it was observed that the monolayer cells were distributed in a radial or spiral form. After digestion, human umbilical cord mesenchymal stem cells (hUC-MSCs) appeared as spherical single cells floating in the culture medium. After centrifugation, they were passaged again at a ratio of 1:3. After 12 hours of passage, most cells began to adhere. On the third day of passage, the cell fusion rate reached 80%-90%, and the cells showed a radial distribution. The detailed distribution morphology can be referred to. Figure 3 After 4 passages, hUC-MSCs still maintained a strong proliferation capacity.

[0111] (3) Identification of hUC-MSCs differentiation into osteoblasts, adipocytes, and chondrocytes

[0112] Human umbilical cord mesenchymal stem cells (hUC-MSCs) can differentiate into multiple directional cells, including osteoblasts, adipocytes, and chondrocytes. The differentiation process can be identified by specific staining methods.

[0113] Test results such as Figure 4 As shown, under osteogenic induction conditions, hUC-MSCs differentiated toward osteoblasts. After 18 days of induction and culture, the formed mineralized nodules stained red with Alizarin Red, indicating successful differentiation. Under adipogenic induction conditions, hUC-MSCs differentiated toward adipocytes. After 20 days of induction and culture, the intracellular lipid droplets appeared orange-red with Oil Red O staining, indicating successful differentiation. Under chondrogenic induction conditions, hUC-MSCs differentiated toward chondroblasts. After 20 days of induction and culture, the blue areas stained with Alcian Blue represented the acidic mucopolysaccharides in the cartilage tissue, indicating successful differentiation.

[0114] (4) Identification of hUC-MSCs surface markers

[0115] According to the identification criteria established by the International Society for Cellular Therapy, mesenchymal stem cells exhibit high expression (≥99%) of positive markers (CD90, CD73, and CD105) and low expression (≤1%) of negative markers (CD34, CD45, and HLA-DR). Flow cytometry analysis showed that CD90 expression was 99.7%, CD73 expression was 99.8%, and CD105 expression was 99.8%, all showing high expression. However, CD34 expression was 0.67%, CD45 expression was 0.57%, and HLA-DR expression was 0.51%, all showing low expression. Figure 5 The above test results fully prove that these cells meet the mesenchymal stem cell phenotypic standards set by the International Mesenchymal Stem Cell Organization Stem Cell Committee and are suitable for subsequent experiments.

[0116] Example 2. Establishment and evaluation of T2DM rat model, hUC-MSCs and hUC-MSCs+Met treatment of T2DM rat model

[0117] (1) Experimental methods

[0118] (1) SD rats

[0119] Male Sprague-Dawley rats, 8 weeks old, weighing 180-220 g, were specific pathogen-free (SPF) and purchased from Hunan Slake Jingda Laboratory Animal Co., Ltd., quality certificate number: SCXK(Xiang)-2019-0004. They were housed at the Laboratory Animal Center, Institute of Medical Sciences, Southwest University, license number: SYXK(Yu)2022-0008, located at the 1st Floor, Sericulture Palace, Southwest University, Tiansheng Road, Beibei District, Chongqing. The experimental environment was maintained at a temperature of 20±5°C and a humidity of 50±10%. They had free access to food and water, with a 12-hour day-night interval. This experiment was approved by the Laboratory Animal Ethics Committee of Southwest University. The use of experimental animals complied with the decisions of the Southwest University Ethics Committee. IACUC number: IACUC-20250224-07. Documents related to animal ethics approval are provided in the Appendix.

[0120] (2) Preparation of main reagents and feed required for rats

[0121] 1) Citric Acid Buffer / Sodium Citrate Buffer: Weigh 2.1 g of citric acid (molecular weight: 210.14) and dissolve it in 100 ml of ultrapure water to prepare Solution A. Weigh 2.94 g of sodium citrate (molecular weight: 294.1) and dissolve it in 100 ml of ultrapure water to prepare Solution B. Before the intraperitoneal injection of STZ, mix Solution A and Solution B in a 1:1 ratio and adjust the pH to 4.2-4.5 to prepare a mixed solution AB for later use.

[0122] 2) STZ Solution: Weigh STZ powder and dispense the required amount of STZ at 25 mg / kg based on the total body weight of the model rat. Dissolve the STZ powder in the AB mixture at a concentration of 10 mg / ml to prepare the STZ solution. Protect the STZ solution from light and store on ice. Inject intraperitoneally within 30 minutes of preparation.

[0123] 3) 1× PBS (0.01M Phosphate Buffer): 1 L of PBS phosphate buffer (powder) was purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd. Prepare by dissolving in 1 L of distilled water to a concentration of 0.01 M and a pH of 7.2-7.4. After autoclaving, it can be stored at room temperature for extended periods.

[0124] 4) Met solution: dissolve 1g of metformin hydrochloride sustained-release tablets in 10ml of PBS solution to prepare a 10% Met solution, which is used immediately after preparation.

[0125] 5) High-sugar, high-fat feed formula: 60% basal feed, 20% sucrose, 10% lard, 2% cholesterol, 2% sodium cholate, 6% other additives. To prepare, accurately weigh all ingredients. Heat and melt the lard. Mix the dry ingredients and pour the melted lard into the mixture. Stir continuously until evenly combined. Allow to cool naturally, cut into pieces, package, label, and store in a refrigerator at 4°C.

[0126] (3) Establishment and grouping of T2DM rat model

[0127] Thirty six-week-old male Sprague-Dawley rats were acclimated for one week. Before the experiment, normoglycemia was confirmed in all rats. The rats were randomly divided into two groups: 10 rats were randomly selected and fed a normal chow diet (NCD), designated the NCD group; the remaining 20 rats were fed a high-fat diet (HFD), designated the HFD group. The rats were weighed weekly. After four weeks of feeding on the HFD, the rats in the HFD group were intraperitoneally injected with 1% STZ at a dose of 25 mg / kg (designated the HFD+STZ group). The control group received an equal volume of citrate buffer. Food and water were promptly replenished after the injections. On the third day after STZ injection, tail vein blood glucose was measured every other day. Random blood glucose levels exceeded 16.7 mmol / L for three consecutive times and were measured twice weekly after one week of model establishment. Successful model establishment was considered successful when all four random blood glucose levels remained above 16.7 mmol / L, and glucose tolerance and insulin tolerance tests demonstrated IR.

[0128] (4) Stem cell therapy and metformin treatment

[0129] T2DM rats were divided into T2DM group, Met group, MSC group, MSC+Met group, and NCD group, with 6 rats in each group (n=6). The normal control rats in the NCD group were the NC group; T2DM group (each rat was injected with an equal amount of PBS through the tail vein); Met group (the rats were gavaged with 200 mg / kg once a day for 4 weeks); MSC group (1×10 6 hUC-MSCs were suspended in 200 μL PBS and injected through the tail vein); MSC+Met group (1×10 6 After the rats were treated with hUC-MSCs, they were gavaged with Met solution at a dose of 200 mg / kg once a day for 4 weeks. After treatment, the blood glucose levels in the tail vein of the T2DM group, Met group, MSC group, and MSC+Met group were continuously monitored.

[0130] (5) Database analysis

[0131] The online NCBI-GEO database was used to analyze and screen the differentially expressed genes between the liver transcriptomes of type 2 diabetic rats and normal rats, and signal pathway enrichment analysis was performed on the differentially expressed genes.

[0132] (6) Statistics and Analysis

[0133] All experiments were performed at least three times independently. The quantitative data are expressed as mean ± SD. All data were subjected to t-test, and the significance was calculated within the 95% confidence interval. When P < 0.05, the value was considered statistically significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0134] (2) Experimental results

[0135] (1) Effects of high-sugar and high-fat diet combined with STZ on physiological indicators of SD rats

[0136] The initial body weights of the NCD group and the HFD group were 175.2±10.3g and 179.3±12.7g, respectively, with no statistically significant difference between the groups (P>0.05). After 4 weeks, the body weights of the rats in the NCD group were 242.8±9.8g, and those in the HFD group were 309.6±12.5g. After 6 weeks, the body weights of the rats in the NCD group were 287.6±8.7g, and those in the HFD group were 275.8±18.6g. These results indicate that a high-sugar and high-fat diet can significantly increase the body weight of rats compared to a normal diet. Furthermore, after STZ injection, the body weights of the rats in the HFD group began to decrease from the fourth week, with significant intra-group differences. (Results as shown in the figure) Figure 6 -A).

[0137] Compared with the NCD group, the blood glucose level of rats in the HFD group increased at 4 weeks, but the difference was small (P>0.05). On the 3rd day after STZ injection, the blood glucose level of rats in the HFD+STZ group reached 16.7±0.8mmol / L (P<0.05). After that, the blood glucose level was stabilized and maintained above 16.7mmol / L. Figure 6 The above results suggest that a high-sugar and high-fat diet combined with a small dose of STZ injection can significantly increase the blood glucose level in rats.

[0138] This study used IPGTT and IPITT methods to examine the effects of a high-sugar and high-fat diet combined with low-dose STZ injection on glucose tolerance and insulin tolerance in SD rats. Figure 6 As shown in Figure 3-C, compared with the NCD group, the blood glucose level of rats in the HFD+STZ group increased rapidly (P<0.001), while the rate of decrease was relatively slow, indicating that the rats had impaired glucose tolerance. Figure 6As shown in Figure 3-D, blood glucose levels in the NCD group decreased rapidly after insulin administration; blood glucose levels in the HFD+STZ group gradually decreased after insulin administration, but the blood glucose concentrations were significantly higher than those in the NCD group (P<0.001), suggesting that the HFD+STZ group developed insulin resistance. Both results showed that at all time points after injection, the extent of blood glucose reduction in the HFD group was significantly lower than that in the NCD group, indicating a significant decrease in insulin sensitivity in the HFD group. Taken together, these results indicate that the HFD combined with intraperitoneal injection of a low-dose STZ used in this experiment induced elevated blood glucose, impaired glucose tolerance, and decreased insulin sensitivity in rats, consistent with characteristic changes in T2DM and demonstrating successful model establishment.

[0139] (2) Database analysis results

[0140] The data for this study were derived from the GEO dataset GSE197097, which is the result of high-throughput RNA sequencing of the livers of normal and type 2 diabetic rats. Through in-depth analysis of the sequencing data, this study screened out differentially expressed genes between the two groups and used KEGG to analyze the enrichment of signaling pathways in the liver. The analysis results are shown in Figure 2. Figure 7 As shown, the MAPK signaling pathway plays a key role in liver signaling pathways and plays a decisive role in the physiological processes of the liver in normal and type 2 diabetic rats. Based on these findings, this study selected the MAPK signaling pathway as the research object, aiming to further explore the mechanism of action of human umbilical cord mesenchymal stem cells in the treatment of type 2 diabetic rats, clarify its synergistic effect with metformin's action site, explore its regulatory effect on key proteins in liver glucose and lipid metabolism, and systematically analyze the regulatory effect of this pathway on inflammatory factors and apoptosis pathways.

[0141] (3) Summary and Analysis

[0142] During the animal model establishment phase, the results showed that a high-sugar, high-fat diet significantly promoted weight gain in rats. After STZ injection, the weight of rats in the HFD group began to decline from the 4th week, and the intra-group difference increased significantly. The combined injection of a high-sugar, high-fat diet and a small dose of STZ can effectively increase the blood sugar level in rats. The results of the glucose tolerance and insulin tolerance test experiments showed that the blood sugar of rats in the HFD+STZ group rose rapidly in the IPGTT experiment and then fell slowly, indicating that their glucose tolerance was significantly reduced; in the IPITT experiment, after intraperitoneal injection of insulin, the blood sugar of rats in the HFD group gradually decreased, but the blood sugar concentration at each time point was significantly higher than that of the NCD group, indicating insulin resistance, and the insulin sensitivity of rats in the HFD group decreased significantly. These experimental results show that the present invention has successfully constructed a type 2 diabetes rat model with T2DM characteristics, and this model provides support for subsequent in-depth research.

[0143] The research data were derived from the GEO dataset GSE197097. In-depth analysis and pathway enrichment of this dataset revealed that the MAPK signaling pathway plays a key role in liver signaling and is the most significant factor influencing the differences in liver physiological processes between normal rats and rats with type 2 diabetes. Based on this key finding, the study identified the MAPK signaling pathway as a key target for future research, providing guidance for subsequent experiments.

[0144] Example 3. Improvement of hepatic glucose and lipid metabolism in T2DM rats by hUC-MSCs combined with Met therapy

[0145] (1) Experimental methods

[0146] (1) Total protein extraction from rat liver tissue

[0147] 1) Prepare protein lysis buffer: 980 μL RIPA lysis buffer, 10 μL PMSF protease inhibitor, and 10 μL phosphatase inhibitor, and mix evenly.

[0148] 2) Add liquid nitrogen and grind the liver tissue thoroughly in a mortar at low temperature. Add liquid nitrogen during the grinding process to keep the liver tissue in a hard solid state. Grind it into a white powder. After grinding, place it into a centrifuge tube, add the prepared protein lysis buffer, and lyse it on ice for 1 hour to 1.5 hours.

[0149] 3) Total protein extraction: Centrifuge the homogenized tissue (4°C, 12,000 g, 5 min). Carefully aspirate the supernatant, taking care not to aspirate the precipitate. The aspirated supernatant is the extracted liver tissue total protein, which will be used for subsequent protein concentration determination.

[0150] (2) Tissue section staining

[0151] Paraffin tissue section staining is a commonly used technique in histological research. This study applies it to liver tissue samples to observe the liver's physiological state and explore physiological changes after treatment. The liver is a key metabolic and detoxification organ, and a variety of factors can affect its function and structure. After fixation, sectioning, and staining, the stained sections are observed under a microscope to compare changes in cell morphology, tissue structure, and protein expression before and after treatment, providing morphological evidence and theoretical support for evaluating treatment efficacy and understanding pathogenesis.

[0152] (3) Tissue dehydration and embedding

[0153] After obtaining rat liver tissue, it was quickly fixed in 4% PFA fixative at 4°C for at least 24 hours to prevent autolysis and decay. Following fixation, the liver tissue was dehydrated using a gradient alcohol dehydration method, progressing from low to high alcohol concentration. The procedure was as follows: the fixed tissue was immersed in 55%, 65%, 75%, and 85% ethanol solutions for 1 hour each. Subsequently, the fixed tissue was dehydrated twice in 95% ethanol for 30 minutes each, and then twice in anhydrous ethanol for 30 minutes each to completely remove moisture from the tissue. Following dehydration, the liver tissue was immersed in 1 / 2 xylene for 30 minutes, followed by an immersion in pure xylene for 1 hour. The immersed liver tissue was placed in a white embedding cassette and placed in an embedding machine for wax immersion at 65°C overnight. After wax immersion, the liver tissue was embedded and numbered. All embedded and numbered tissues were collected and stored at 4°C.

[0154] (4) HE staining

[0155] The HE staining protocol includes the following steps: 1) Place the sections in a 60°C oven for 2 hours. 2) Dewax and hydrate according to the procedure in Table 1. 3) Place the sections, along with the staining rack, in a beaker and slowly rinse under running water to remove the alcohol until the sections are clean and transparent. Stain with hematoxylin for 5 minutes, then rinse several times with tap water. 4) Incubate with 1% hydrochloric acid-ethanol solution for 5 seconds, then rinse with water for 30 seconds. 5) Bluing with 1% ammonia solution for 1 minute, then rinse several times with water. 6) Stain with eosin solution. Sections should first be dehydrated with 80% ethanol before staining with eosin solution (alcohol-soluble) for 8 minutes. 7) Rapidly dehydrate in 95% ethanol I and II for 10 seconds. 8) Dehydrate in anhydrous ethanol I and II for 5 minutes each, then permeabilize in xylene I and II for 10 minutes each. 9) Mount the sections with clear nail polish. Examine under a microscope and photograph.

[0156] Table 1. Dewaxing and hydration procedures for HE staining experiments

[0157] Xylene I 5min Xylene II 5min 1 / 2 xylene 5min 100% ethanol I 5min 100% ethanol II 5min 95% ethanol 5min 85% ethanol 5min 75% ethanol 5min <![CDATA[ddH2O]]> 5min

[0158] (5) PAS staining

[0159] The PAS staining protocol includes the following steps: 1) Place the sections in a 60°C oven for 2 hours; 2) Dewax and rehydrate according to the procedure in Table 2; 3) Remove the periodic acid solution and equilibrate to room temperature. Add 100 μL of the periodic acid solution to each sample, incubate in a humidified chamber in the dark for 10 minutes, then remove the periodic acid solution and wash on a shaker for 5 minutes. 4) Add 100 μL of Schiff's reagent to each sample, place in a humidified chamber, and stain in a 37°C oven in the dark for 1 hour. Remove the staining solution, soak in distilled water, and wash on a shaker for 5 minutes. 5) Add 100 μL of hematoxylin stain to each sample and stain for 30 seconds. Remove the staining solution and rinse in ddH2O for 30 seconds. 6) Rapidly dehydrate in 95% ethanol I and II for 10 seconds. 7) Dehydrate in anhydrous ethanol I and II for 5 minutes each, and permeabilize in xylene I and II for 10 minutes each. 8) Mount the sections with clear nail polish. Examine under a microscope and photograph.

[0160] Table 2. Dewaxing and hydration procedures for PAS staining experiments

[0161]

[0162]

[0163] (6) ELISA experiment

[0164] The ELISA kits used in the experiments of the present invention are shown in Table 3.

[0165] Table 3. ELISA kit information

[0166]

[0167] The ELISA experimental steps are as follows:

[0168] 1) Prepare an appropriate concentration of antibody (usually 1:10,000) and dilute it in carbonate buffer (pH 9.6). Add 100 μL of the diluted antibody solution to each well of a 96-well plate. Incubate at 37°C for 2 h.

[0169] 2) Wash the plate 3-5 times with PBS, using 200 μL / well of washing buffer each time to ensure that all unbound substances in the wells are washed away.

[0170] 3) Add 200 μL of blocking solution (5% skim milk powder solution) to each well and incubate at room temperature for 2 hours to block unbound sites on the well surface and reduce nonspecific binding. After incubation, repeat the plate wash step.

[0171] 4) Dilute the sample or standard antigen to be tested in a gradient manner.

[0172] 5) Add 100 μL of diluted sample or standard to each well and incubate at room temperature for 2 h.

[0173] 6) After incubation, wash the plate 3-5 times. Add 100 μL of diluted detection antibody to each well. Incubate at room temperature for 2 hours, then wash the plate.

[0174] 7) Add 100 μL of diluted enzyme-labeled secondary antibody (enzyme-labeled antibody that binds to the detection antibody) to each well.

[0175] 8) After incubation at room temperature for 1 hour, wash the plate again.

[0176] 9) Add 100 μL of enzyme substrate solution to each well and react for about 5-30 minutes (the specific time depends on the color development) to allow the enzyme to catalyze the substrate reaction to produce color.

[0177] 10) Add 100 μL of 2 M sulfuric acid to terminate the reaction and stop the substrate color development reaction. After termination, the color will turn from blue to yellow.

[0178] 11) Read the absorbance (OD value) of each well at a wavelength of 450 nm on a microplate reader.

[0179] (7) Immunohistochemistry (IHC)

[0180] 1) Place the slices in a 60°C oven and bake for 2 hours.

[0181] 2) Dewax and hydrate according to the procedures shown in Table 4.

[0182] Table 4. Deparaffinization and hydration schedule for IHC experiments

[0183] Xylene 5min Xylene I 5min 1 / 2 xylene 5min 100% ethanol I 5min 100% ethanol II 5min 95% ethanol 5min 85% ethanol 5min 75% ethanol 5min <![CDATA[ddH2O]]> 5min

[0184] 3) After dewaxing, rinse the remaining alcohol with tap water, then wash with double-distilled water for 3 x 5 min. Perforate the nuclear protein with Triton X-100 (0.5%) (0.5% Triton X-100 in PBS) for 2 h. This step can be skipped for membrane proteins.

[0185] 4) Antigen retrieval (microwave retrieval)

[0186] a. Antigen retrieval solution (prepared immediately for use): 1.71 g trisodium citrate / 2H2O, 0.2 g citric acid, and dilute to 500 mL with ddH2O.

[0187] b. Antigen retrieval process: Microwave the antigen retrieval solution for 2 x 5 minutes, bring to a boil, add the slide, microwave for 5 minutes, remove from the microwave and allow to cool at room temperature for 15 minutes, microwave for another 2 minutes, and cool to room temperature.

[0188] 5) Dry the slide, draw a hydrophobic circle on the wax block, and inactivate endogenous peroxidase by incubating at room temperature for 20 minutes. Protect from light and perform the operation in a dark box.

[0189] 6) Wash with PBS for 3×5 min.

[0190] 7) Block with 10% goat serum (in PBS) for 1 h.

[0191] 8) Incubate with primary antibody at 4°C overnight, protecting from light.

[0192] 9) Wash with PBS for 3 x 5 min

[0193] 10) Add reaction enhancement solution (reagent II) dropwise and incubate at room temperature for 20 minutes.

[0194] 11) Wash with PBS for 3 x 5 min

[0195] 12) Add enhanced enzyme-labeled goat anti-rabbit / mouse IgG polymer and incubate at room temperature for 20 minutes.

[0196] 13) Wash with PBS for 3 x 5 min

[0197] 14) DAB color development (add 50 μl DAB concentrate to 1 ml of base solution), mix well, and develop color for 8 minutes.

[0198] 15) After color development, wash with ddH2O on a shaker for 5 minutes.

[0199] 16) Counterstain with hematoxylin for 5 minutes.

[0200] 17) Differentiate with 1% hydrochloric acid ethanol solution for 7 seconds, rinse with tap water, and return to blue with 1% ammonia solution for 1 minute.

[0201] 18) Dehydration: Dehydrate according to the steps shown in Table 5.

[0202] Table 5

[0203]

[0204]

[0205] 19) Use transparent nail polish to seal the slides. Examine under a microscope and take photos.

[0206] (8)TUNEL

[0207] The TUNEL experimental steps include: 1) Place the slices in a 60℃ oven and bake for 2 hours. 2) Dewax and hydrate according to the procedure shown in Table 6. 3) Dry the slides and draw a hydrophobic circle on the wax block. 4) Dilute the 2mg / mL ProteinaseK solution with PBS to a final concentration of 40μg / mL at a ratio of 1:50, add 100μL to each sample, and incubate at 37℃ for 30min. 5) Immerse the slice sample in 1×PBS and wash for 3×5min, and absorb the excess liquid with absorbent paper. 6) Prepare TUNEL reaction solution (prepare for immediate use): 10μL of TdT enzyme, 488TUNEL Reaction Buffer 240μL, total reaction volume 250μL. 7) Add 50μL TUNEL reaction solution to each sample and incubate at 37°C in the dark for 1 hour. 8) Discard the TUNEL reaction solution, wash twice with PBS, and then wash 3×5 minutes with 0.1% TritonX-100 (prepared in PBS, containing 5mg / mL BSA). 9) Add 50μL DAPI staining solution to each sample and incubate at room temperature in the dark for 5 minutes. After staining, discard the DAPI staining solution and wash 2×5 minutes with PBS. 10) Use filter paper to absorb excess liquid, add 100μL PBS to the sample area to keep the sample moist, and observe immediately under a fluorescence microscope.

[0208] Table 6. Dewaxing and hydration procedures for TUNEL assays

[0209] Xylene I 5min Xylene II 5min 1 / 2 xylene 5min 100% ethanol I 5min 100% ethanol II 5min 95% ethanol 5min 85% ethanol 5min 75% ethanol 5min <![CDATA[ddH2O]]> 5min

[0210] (2) Experimental results

[0211] (1) Effects of hUC-MSCs and combined treatment on physiological parameters of T2DM rats

[0212] T2DM is a complex metabolic disease, the pathogenic mechanism of which involves multiple aspects such as insulin resistance and pancreatic β-cell dysfunction. These factors interact with each other, resulting in an imbalance in energy metabolism in the affected individuals, which in turn presents the typical characteristics of progressive weight loss. In this experiment, after treatment with hUC-MSCs and hUC-MSCs+Met, the weight of rats was restored to a certain extent. Figure 8 As shown in Figure 2A, the weight of rats in the T2DM group continued to decrease, while the weight of rats in the Met group continued to increase but did not return to the level of the NC group. After increasing in weight, rats in the MSC and MSC+Met groups exceeded those in the NC group on day 4 of treatment, with the MSC+Met group showing a greater weight gain than the MSC group. Following treatment, the mental state of rats in the MSC and MSC+Met groups recovered, and their weight gradually increased. This weight gain can be considered an outward sign of recovery.

[0213] In the management and treatment of T2DM, blood glucose control plays a core role, and its effectiveness is directly related to the progression of the disease and the quality of life of patients. This study determined the effect of hUC-MSCs and hUC-MSCs+Met treatment on the blood glucose recovery of T2DM rats by measuring the blood glucose level in the tail vein of rats. Figure 8 As shown in Figure 2-B, blood glucose levels in T2DM rats decreased to a certain extent after treatment with hUC-MSCs and hUC-MSCs + Met. In contrast, blood glucose levels in rats in the T2DM group continued to rise. This comparison further supports the scientific validity of this experiment and confirms the role of hUC-MSCs and hUC-MSCs + Met in lowering blood glucose levels in T2DM rats. The blood glucose levels in the Met group decreased less than those in the MSC and MSC + Met groups, with the MSC + Met group achieving the greatest blood glucose recovery. This indicates that combined hUC-MSCs + Met therapy can effectively lower blood glucose levels in T2DM rats, consistent with the results of controlling insulin resistance.

[0214] As important means of evaluating the body's glucose metabolism function, the intermittent glucose tolerance test (IPGTT) and the insulin tolerance test (IPITT) can accurately reflect the body's ability to regulate blood sugar fluctuations, and thus intuitively display the level of insulin secretion and metabolic balance. These two tolerance tests were conducted to confirm the restorative effect of hUC-MSCs and hUC-MSCs+Met treatment on the blood sugar regulation ability of T2DM rats, thereby reflecting the degree of improvement of the treatment on the body health of T2DM rats. The results are as follows Figure 8 -C. Figure 8 -D shows that after the injection of glucose / insulin solution, the blood glucose of each group reached the highest level at 30 minutes. Among them, the blood glucose of the NC group rose more slowly, and the subsequent recovery trend was stable, indicating that the body always maintained a healthy state and could effectively buffer the impact of external factors on the overall blood glucose level. The blood glucose of the T2DM group rose the fastest and fell rapidly, with a large fluctuation range, and could not drop to the initial blood glucose level, which fully demonstrated that it had lost the ability to regulate blood glucose. In comparison, the blood glucose regulation ability of the Met group, MSC group, and MSC+Met group was restored, among which the recovery effect of the MSC+Met group was the most significant, followed by the MSC group. Among all the treatment groups, the blood glucose of the MSC+Met group rose the slowest and was able to drop below the initial level. In summary, hUC-MSCs combined with Met treatment can significantly restore the blood glucose regulation ability of T2DM rats.

[0215] (2) hUC-MSCs and combined treatment can improve liver function, dyslipidemia and inhibit inflammation in T2DM rats

[0216] Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) are key enzymes in liver cells, playing an important catalytic role in the metabolism of amino acids. Their serum levels are often used as key indicators for evaluating liver function. Under normal physiological conditions, the levels of these two enzymes in serum are maintained at a relatively stable low level. However, during the progression of T2DM, multiple pathological factors such as hyperglycemia, insulin resistance, and oxidative stress work together to damage liver cells and increase cell membrane permeability, thereby releasing AST and ALT in liver cells into the blood, causing a significant increase in the levels of these two enzymes in serum. Liver function test results are as follows: Figure 9 As shown in the figure, the AST and ALT levels in the serum of rats in the T2DM group were significantly higher than those in other groups, reaching the highest value, which intuitively reflects that T2DM caused more serious damage to the rat liver. The AST and ALT levels in the serum of rats in the Met group and MSC group were reduced to a certain extent compared with the T2DM group, indicating that these two treatment methods have played a repairing role in liver damage to a certain extent. It is worth noting that the AST and ALT levels in the serum of rats in the MSC+Met group are closest to those in the normal group, which fully demonstrates that hUC-MSCs combined with Met treatment has significant advantages in improving liver function. The changes in these two indicators effectively show that hUC-MSCs and hUC-MSCs+Met treatment can effectively improve the body's liver metabolism level and liver function repair ability, and have a good alleviating and repairing effect on liver damage caused by T2DM.

[0217] High-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) are key indicators of serum lipid metabolism and play an extremely important role in the body's lipid transport and metabolic homeostasis. Figure 10As shown, serum HDL-C levels in T2DM rats were significantly lower than those in the normal control group, while LDL-C levels were significantly higher, reaching extreme values ​​among all experimental groups. This result clearly demonstrates that T2DM causes severe disruptions in rat lipid metabolism and indirectly indicates that T2DM significantly impairs the liver's function in regulating lipid metabolism. In sharp contrast, serum HDL-C levels in the Met and MSC groups increased to varying degrees compared with those in the T2DM group, while LDL-C levels decreased. This demonstrates that these two treatments repaired and regulated the impaired liver's lipid metabolism to a certain extent, effectively improving the body's lipid metabolism disorders. Most notably, serum HDL-C and LDL-C levels in the MSC+Met group were closest to those in the normal control group, with HDL-C returning to near-normal levels and LDL-C decreasing to a range not significantly different from those in the normal control group. This result strongly demonstrates that hUC-MSCs combined with Met therapy has outstanding advantages in improving liver lipid metabolism function, can more effectively reshape the liver's normal regulatory mechanism of lipid metabolism, and restore the balanced levels of HDL-C and LDL-C in serum. Taking into account the dynamic changes of the above two indicators, it can be clearly concluded that hUC-MSCs and hUC-MSCs + Met treatment can effectively improve the body's liver metabolism level and significantly enhance the liver's ability to regulate and repair lipid metabolism. It has a good effect on alleviating and repairing liver lipid metabolism disorders and related liver damage caused by T2DM, providing a highly promising intervention strategy for the treatment of abnormal lipid metabolism and liver lesions in T2DM patients.

[0218] Interleukin-10 (IL-10), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) are important cytokines in the body that play a key role in immune regulation and inflammatory response. Their serum levels are important indicators for measuring the body's inflammatory state. Under normal physiological conditions, the body's immunity is in a balanced steady state. IL-10 is maintained at a moderate level, playing an anti-inflammatory role and inhibiting the excessive activation of inflammatory cells; IL-6 and TNF-α exist at lower levels and participate in physiological processes such as normal immune defense and tissue repair. However, in the course of type 2 diabetes mellitus (T2DM), factors such as long-term hyperglycemia, insulin resistance, and oxidative stress work together to disrupt the balance between the body's immune and inflammatory regulation. Hyperglycemia promotes immune cell dysfunction, insulin resistance interferes with intracellular signal transduction pathways, and oxidative stress produces a large number of free radicals. These factors stimulate inflammatory cells to secrete large amounts of IL-6 and TNF-α, while inhibiting the production of IL-10, resulting in a significant increase in serum IL-6 and TNF-α levels, while a significant decrease in IL-10 levels, resulting in an overall state of hyperinflammatory response. Figure 11 As shown, serum IL-6 and TNF-α levels in rats in the T2DM group were significantly higher than those in the other groups, reaching their highest values, while IL-10 levels were significantly lower than those in the other groups. This clearly reflects that T2DM leads to an imbalance in the rats' inflammatory response, resulting in a highly inflammatory state. Serum IL-6 and TNF-α levels in rats in the Met and MSC groups were both reduced compared to those in the T2DM group, while IL-10 levels were increased, indicating that both treatments modulated the inflammatory response to a certain extent and improved the inflammatory imbalance. Notably, serum IL-6, TNF-α, and IL-10 levels in rats in the MSC+Met group were closest to those in the normal control group, demonstrating the significant advantage of hUC-MSCs combined with Met therapy in regulating immune-inflammatory balance. These changes in these three indicators strongly suggest that hUC-MSCs and hUC-MSCs+Met therapy can effectively regulate immune-inflammatory levels and alleviate and repair the immune-inflammatory disorder caused by T2DM.

[0219] (3) Study on liver pathological characteristics of hUC-MSCs and combined therapy based on HE, PAS and immunohistochemistry

[0220] HE staining results Figure 12 The liver cells in the NC group were regular in morphology, with orderly arrangement of liver cells, normal nuclei, uniform cytoplasm staining, and clear liver lobule structure, showing the typical morphology of healthy liver tissue, indicating that the liver tissue structure of normal rats is intact and the cell function is normal. Figure 12 -A. The liver tissue of rats in the T2DM group showed significant changes compared to the NC group. Irregular cell morphology, disordered hepatocyte arrangement, enlarged intercellular spaces in some areas, and possible cell swelling or fatty degeneration. There were obvious red areas in some areas, or pathological changes such as inflammatory cell infiltration or hemorrhage were indicated. Figure 12 -B. The liver tissue damage in the Met group was improved compared with that in the T2DM group, and the cells were arranged more orderly. However, some cells still had abnormal morphology, and the integrity of the tissue structure was not fully restored. This indicates that metformin treatment can alleviate liver damage to a certain extent, but the effect is limited. For details, see Figure 12 -C. The cell morphology and arrangement of the liver tissue of rats in the MSC group were better than those in the T2DM group, and the red abnormal area was reduced, but there was still a difference compared with the NC group, indicating that human umbilical cord mesenchymal stem cell treatment has a repair effect on liver damage in T2DM rats, but single use cannot completely restore liver tissue to normal. For details, see Figure 12-D. The cell morphology and arrangement of the liver tissue in the MSC+Met group were closer to those in the NC group, the abnormal red area was further reduced, and the cell structure was clearer. This indicates that human umbilical cord mesenchymal stem cells combined with metformin treatment have a better effect on repairing liver tissue damage in T2DM rats and are more conducive to restoring the normal structure of liver tissue. For details, see Figure 12 -E. These results indicate that hUC-MSCs and hUC-MSCs+Met treatment can improve liver tissue damage in T2DM rats.

[0221] The results of PAS staining were as follows Figure 13 The liver cells in the NC group were regular in morphology and orderly in arrangement. After PAS staining, glycogen deposition was evenly distributed with moderate staining intensity, showing the normal glycogen deposition in healthy liver tissue. This indicates that the liver of normal rats has normal function in glycogen metabolism, intact tissue structure, and can maintain the reasonable storage and metabolism of glycogen. Figure 13 -A. The liver tissue of rats in the T2DM group showed significant changes compared to the NC group. The cells were arranged in disorder, and glycogen deposition increased significantly, with the staining showing a darker purple-red color. This indicates excessive glycogen deposition in the liver tissue under T2DM conditions. This may be due to the disorder of glucose metabolism caused by diabetes, which leads to an imbalance between glycogen synthesis and decomposition, thereby affecting the normal function of the liver. For details, see Figure 13 -B. Glycogen deposition in the liver tissue of rats in the Met group was improved compared with that in the T2DM group. The purple-red color became lighter and the amount of glycogen deposition decreased. However, compared with the NC group, a certain degree of glycogen deposition still existed. This indicates that metformin treatment regulated liver glucose metabolism to a certain extent and alleviated excessive glycogen deposition, but it did not restore glycogen deposition to normal levels. For details, see Figure 13 -C. After PAS staining of the liver tissue of rats in the MSC group, the amount of glycogen deposition was less than that of the T2DM group, and the purple-red staining intensity was weaker. However, there was still a gap compared with the NC group. This shows that human umbilical cord mesenchymal stem cell treatment has a certain repair effect on the glycogen metabolism disorder in the liver of T2DM rats and can reduce excessive glycogen deposition. However, it cannot completely correct the glycogen metabolism abnormality when used alone. For details, see Figure 13 -D. The glycogen deposition in the liver tissue of rats in the MSC+Met group was closest to that in the NC group. The purple-red color of the staining was significantly lighter, and the amount of glycogen deposition was greatly reduced. This indicates that human umbilical cord mesenchymal stem cells combined with metformin treatment can effectively improve the glucose metabolism in the liver of T2DM rats, significantly reduce excessive glycogen deposition, and is more conducive to restoring the normal function of liver tissue in glycogen metabolism. For details, see Figure 13 -E. Glycogen deposition positive rate results are as follows Figure 13As shown in Figure 3-F, the positive rate in the T2DM group was significantly higher than that in the NC group, while the positive rates in the Met group, MSC group, and MSC+Met group decreased in order, with the MSC+Met group being closest to the NC group. This result further demonstrates that hUC-MSCs and hUC-MSCs+Met treatment can effectively improve glycogen deposition in the liver tissue of T2DM rats and alleviate glucose metabolism disorders.

[0222] (4) hUC-MSCs and hUC-MSCs+Met treatment can improve hepatic glucose and lipid metabolism in T2DM rats

[0223] IHC staining results Figure 14 and Figure 15 As shown. In the liver tissue of rats in the NC group, the expression level of ICAM-1 protein was extremely low, with a faint, light staining and a sparse distribution, indicating that the inflammatory-related signaling pathways in normal liver tissue are in a dormant state, without obvious inflammatory activation, creating a stable internal environment for glucolipid metabolism. The expression of ICAM-1 protein in the liver tissue of rats in the T2DM group increased significantly, with a deep staining color and a widespread and dense distribution, indicating that the liver inflammatory response is strongly activated under T2DM conditions, with a large amount of inflammatory factors released, which seriously interferes with the signal transduction and regulatory network of glucolipid metabolism. The expression of ICAM-1 protein in the liver tissue of rats in the Met group was significantly lower than that in the T2DM group, with a lighter staining degree and a narrowed distribution range, but it was still higher than that in the NC group, indicating that metformin treatment can partially inhibit the inflammatory response and reduce the adverse effects of inflammation on glucolipid metabolism, but it has not yet returned the inflammatory level to normal. In the liver tissue of rats in the MSC group, ICAM-1 protein expression was significantly lower than that in the T2DM group, with lighter staining and a reduced distribution area. However, there was still a difference compared to the NC group, indicating that human umbilical cord mesenchymal stem cell therapy can effectively inhibit liver inflammation and alleviate the interference of inflammation on glucolipid metabolism. However, when used alone, it cannot completely eliminate the hidden dangers of inflammation. In the liver tissue of rats in the MSC+Met group, ICAM-1 protein expression was very similar to that in the NC group, with weak staining and sparse distribution. The inflammatory response was greatly suppressed and basically returned to normal levels, creating a favorable microenvironment for glucolipid metabolism.

[0224] In the liver tissue of rats in the NC group, CPT1A protein expression was abundant and uniform, with clear, moderate staining. This indicates that key enzymes in hepatic fatty acid metabolism are functioning at normal levels, ensuring smooth lipid metabolism processes such as fatty acid β-oxidation, and demonstrating the efficient and orderly function of normal liver lipid metabolism. In the liver tissue of rats in the T2DM group, CPT1A protein expression was significantly reduced, with faint staining and scattered distribution. This indicates that the key rate-limiting step of fatty acid β-oxidation is blocked, preventing fatty acids from entering the mitochondria for metabolism and accumulating in the liver, exacerbating lipid metabolism disorders and leading to pathological changes such as hepatic steatosis. In the liver tissue of rats in the Met group, CPT1A protein expression rebounded, with deeper staining and increased distribution, but still decreased compared to the NC group. This suggests that metformin can promote the expression of proteins related to fatty acid metabolism to some extent and improve lipid metabolism imbalance, but it cannot completely restore normal metabolic levels. In the MSC group, CPT1A protein expression increased in liver tissue, with improved staining and distribution, but not reaching the levels seen in the NC group. This suggests that human umbilical cord mesenchymal stem cells have a certain repairing effect on damaged liver fatty acid metabolism, increasing CPT1A protein expression and promoting fatty acid β-oxidation, but the effect of single treatment is limited. In the MSC+Met group, CPT1A protein expression in liver tissue was abundant and evenly distributed, with staining clarity comparable to that in the NC group. This indicates that the function of key enzymes in fatty acid β-oxidation has essentially returned to normal, effectively repairing key links in glucose and lipid metabolism, and that liver tissue is approaching normal function in terms of glucose and lipid metabolism.

[0225] In summary, the detection and analysis of ICAM-1 and CPT1A protein expression showed that hUC-MSCs and hUC-MSCs+Met treatment could significantly improve the glucose and lipid metabolism status of liver tissue in T2DM rats, and the combined treatment had obvious advantages in restoring the normal metabolic function of the liver.

[0226] (5) hUC-MSCs and combined therapy can improve liver function in T2DM rats through MAPK signaling pathway

[0227] In patients with T2DM, the liver is not only a key target organ for insulin resistance (IR), but also a core site for glucose metabolism. In order to deeply explore the relationship between the molecular mechanism of hUC-MSCs and hUC-MSCs+Met in treating T2DM rats and the MAPK signaling pathway and key proteins in glucose and lipid metabolism, the present invention used Western blot technology to detect the expression of related proteins in rat liver tissue. The results are as follows Figure 16 As shown in Figure 2, after treatment with hUC-MSCs and hUC-MSCs+Met, the expression levels of P-p38MAPK, P-ERK1 / 2, PPARA, AMPKα, and HK2 proteins in rat liver tissue increased significantly (P < 0.05) ( Figure 16 -D~ Figure 16-K), while p38MAPK protein expression remained stable across all groups, with no significant changes. Meanwhile, JNK and SREBF1 protein expression decreased significantly (P < 0.05). P-p38MAPK, the phosphorylated activated form of p38MAPK, plays a key role in cellular stress and inflammatory signaling. P-ERK1 / 2 participates in signal transduction in various physiological processes, including cell proliferation and differentiation. PPARA plays a key role in regulating fatty acid metabolism and energy balance. AMPKα is an important sensor and regulator of cellular energy status, promoting cellular glucose uptake and utilization. HK2, a key enzyme in glycolysis, participates in the phosphorylation of glucose. JNK plays an important regulatory role in cellular stress, apoptosis, and inflammatory response signaling pathways. SREBF1 is primarily involved in the regulation of adipogenesis.

[0228] The above results suggest that hUC-MSCs and hUC-MSCs+Met treatment may reshape the signal transduction network and metabolic balance in the liver tissue of T2DM rats by regulating the expression of proteins related to the MAPK signaling pathway and key proteins in glucose and lipid metabolism, thereby enhancing the liver's sensitivity to insulin and improving the disordered glucose and lipid metabolism.

[0229] like Figure 16 As shown in Figure 3-C, the livers of rats in the NC group showed normal morphology, while those in the T2DM group showed edema, a rough surface, and a lighter color. After treatment with hUC-MSCs and hUC-MSCs+Met, liver edema improved, and the morphology resembled that of the NC group. Overall, hUC-MSCs and hUC-MSCs+Met treatment can improve liver pathology in T2DM rats by regulating the expression of proteins related to the MAPK signaling pathway and glucolipid metabolism.

[0230] (6) hUC-MSCs and combined treatment can regulate the expression of apoptotic proteins in the liver of T2DM rats

[0231] In order to further explore the molecular mechanism of hUC-MSCs and hUC-MSCs+Met in treating T2DM rats and improving their liver apoptosis level, Western blot technology was used to detect the expression of proteins closely related to cell apoptosis in rat liver tissue. The results are as follows Figure 17 After treatment with hUC-MSCs and hUC-MSCs+Met, the expression of Bcl-2 protein in the liver tissue of T2DM rats was significantly increased (P<0.05). Figure 17 -C. Bcl-2 is an anti-apoptotic protein that can inhibit the release of cytochrome C from mitochondria, thereby preventing the occurrence of cell apoptosis. In contrast, the expression levels of Bax and caspase3 proteins decreased significantly (P < 0.05). Figure 17 -B and Figure 17 -D. Bax is a pro-apoptotic protein that can form heterodimers with Bcl-2. When Bax expression increases, it disrupts the balance between pro-apoptotic and anti-apoptotic proteins in the cell, prompting the cell to undergo apoptosis. Caspase-3 is a key protease in the apoptosis process; increased activity and expression of caspase-3 accelerate the apoptosis process.

[0232] The above results indicate that hUC-MSCs and hUC-MSCs+Met treatment may regulate the expression of apoptosis-related proteins such as Bax, Bcl-2, and caspase3, reshape the apoptosis regulatory network in the liver cells of T2DM rats, inhibit excessive apoptosis of liver cells, thereby improving the apoptosis level of the liver of T2DM rats and helping the liver restore normal physiological function.

[0233] (7) hUC-MSCs and combined treatment can inhibit liver apoptosis in T2DM rats

[0234] To further explore the mechanism of hUC-MSCs and hUC-MSCs+Met treatment on liver cell apoptosis in T2DM rats, we used TUNEL (deoxyribonucleotidyl transferase-mediated nick end labeling) technology to detect cell apoptosis in rat liver tissue. TUNEL technology can specifically mark the 3'-OH end produced by DNA breakage in apoptotic cells, and its expression level can intuitively reflect the degree of cell apoptosis. The test results are as follows Figure 18 As shown in the results, after treatment with hUC-MSCs and hUC-MSCs+Met, the number of TUNEL-positive cells in rat liver tissue decreased significantly, indicating a significant decrease in TUNEL expression. This result indicates that hUC-MSCs and hUC-MSCs+Met can effectively reduce DNA fragmentation in liver cells, thereby reducing TUNEL expression and inhibiting cell apoptosis, ultimately improving the liver pathology of T2DM rats and promoting the recovery of liver function.

[0235] (3) Summary and Discussion

[0236] This example investigates the effects of hUC-MSCs and hUC-MSCs+Met treatment on the livers of T2DM rats. The results demonstrate that the combined treatment is highly effective in multiple areas. In terms of physiological indicators, treatment resulted in weight recovery and lowered blood sugar in the rats, significantly restoring their ability to regulate blood sugar, with the MSC+Met group achieving the best results. In terms of liver function, the combined treatment significantly reduced AST and ALT levels, effectively improving liver function. In terms of lipid metabolism, the treatment normalized HDL-C and LDL-C levels, regulating lipid metabolism. In terms of inflammation regulation, the treatment significantly modulated IL-6, TNF-α, and IL-10 levels, balancing immune inflammation. In terms of pathological features, HE staining revealed significant improvement in liver tissue damage, PAS staining indicated reduced glycogen deposition, and immunohistochemistry confirmed optimized lipid metabolism. At the molecular mechanistic level, the combined treatment modulated the MAPK signaling pathway and the expression of key proteins in lipid metabolism. It also inhibited liver cell apoptosis, increased Bcl-2 protein expression, and reduced Bax and caspase3 protein expression and the number of TUNEL-positive cells. In summary, the present invention reveals the therapeutic effect and potential mechanism of hUC-MSCs combined with Met in the treatment of T2DM rats through multiple experiments, opening up a new path for T2DM treatment research.

Claims

1. Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of drugs for the treatment of diabetes.

2. The use according to claim 1, characterized in that The diabetes is type 2 diabetes.

3. The use according to claim 1, characterized in that The human umbilical cord mesenchymal stem cells highly express CD90, CD73 and CD105, and lowly express CD34, CD45 and HLA-DR.

4. The use according to claim 1, characterized in that The preparation method of human umbilical cord mesenchymal stem cells comprises: taking neonatal umbilical cord tissue, digesting it with collagenase to obtain a cell precipitate; adding cell culture medium to the cell precipitate for culturing to obtain human umbilical cord mesenchymal stem cells.

5. Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of drugs for lowering blood sugar and / or restoring blood sugar regulation ability.

6. Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a drug for treating liver damage, liver lipid metabolism disorders, liver glucose metabolism disorders and / or immune inflammatory disorders caused by type 2 diabetes.

7. The use according to claim 6, characterized in that The drugs include: drugs that lower abnormally elevated AST and ALT levels in serum, drugs that increase HDL-C levels in the liver, drugs that lower LDL-C levels in the liver, drugs that lower IL-6 and TNF-α levels in serum, drugs that increase IL-10 levels in serum, drugs that lower excessive liver glycogen deposition, drugs that lower ICAM-1 protein expression in the liver and / or drugs that increase CPT1A protein expression in the liver.

8. Use of human umbilical cord mesenchymal stem cells combined with metformin in the preparation of a drug for inhibiting abnormal apoptosis of liver cells caused by type 2 diabetes.

9. The use according to claim 8, characterized in that The drugs include: drugs that increase the expression of Bcl-2 protein in the liver, and drugs that decrease the expression of Bax and caspase3 proteins in the liver.

10. A pharmaceutical composition for treating type 2 diabetes and its complications, characterized in that: The composition consists of metformin and human umbilical cord mesenchymal stem cells; the complications include any one or more of hyperglycemia, diabetic liver damage, liver glucose and lipid metabolism disorder, and immune inflammatory disorder.

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