Application of dendrobium officinale polysaccharide in preparation of PGC-1alpha signal channel inhibitor

Abstract

The invention discloses application of dendrobium officinale polysaccharide in preparation of a PGC-1alpha signal channel inhibitor, and research shows that dendrobium officinale can regulate and control metabolic gene expression of macrophages in a tumor microenvironment, promote polarization of macrophages M1 and effectively inhibit occurrence and development of tumors; meanwhile, the dendrobium candidum polysaccharide can also promote glycolysis metabolism of tumor-related macrophages, remarkably promote expression of macrophage glycolysis-related genes, inhibit expression of oxidative phosphorylation genes and PGC-1alpha genes, effectively reduce the volume and size of tumors, inhibit PGC-1alpha signal channels in the macrophages and inhibit the growth of tumor cells. In addition, the dendrobium officinale polysaccharide can be used as a PGC-1alpha signal channel inhibitor for preparing antitumor drugs, and a new way is provided for improving the metabolic state in a tumor microenvironment and tumor immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology. More specifically, it relates to the application of Dendrobium officinale polysaccharide in the preparation of PGC-1α signaling pathway inhibitors. Background Technology

[0002] Peroxisome proliferator-inhibitory receptor gamma co-repressor 1α (PGC-1α) is an important transcriptional co-repressor that regulates multiple metabolic pathways. It is primarily expressed in tissues such as the liver, muscle, and brain, and plays a role in regulating cellular energy metabolism and mitochondrial function. It also participates in the regulation of biological processes such as glucose metabolism, fatty acid oxidation, and tumorigenesis and development. PGC-1α plays a crucial regulatory role in maintaining mitochondrial function, regulating energy metabolism, and adapting to different metabolic demands.

[0003] In immune cells, when PGC-1α is inhibited, it can induce the expression of specific genes. These genes stimulate mitochondria in tissues to induce oxidative metabolic responses, playing a crucial regulatory role in the normal metabolic function of macrophages. Studies have found that tumor-infiltrating macrophages exhibit progressively enhanced PGC-1α gene expression, which can impair T cell specific recognition and weaken the body's anti-tumor immunity. Furthermore, the dynamic balance of mitochondrial function and energy regulated by PGC-1α is disrupted, promoting PGC-1α expression in the tumor microenvironment and enhancing the macrophage M2 phenotype. Inhibiting the PGC-1α signaling pathway can promote the macrophage M1 phenotype. Therefore, regulating the PGC-1α signaling pathway in the tumor microenvironment is beneficial for regulating macrophage M1 polarization and inhibiting tumor occurrence and development. While most anti-tumor drugs in existing research have anti-tumor and immunomodulatory effects, there is still a lack of inhibitors or products that can regulate the PGC-1α signaling pathway. Since PGC-1α is associated with a variety of metabolic diseases, there are currently no reports on the regulation and expression of PGC-1α signaling in macrophages. Therefore, there is an urgent need to provide more therapeutic drugs that can effectively regulate the PGC-1α pathway and to provide new avenues for the treatment of metabolic diseases and tumor immunotherapy. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to overcome the shortcomings of existing inhibitors or products for regulating the PGC-1α signaling pathway and to provide the application of Dendrobium officinale polysaccharide in the preparation of PGC-1α signaling pathway inhibitors.

[0005] The purpose of this invention is to provide new applications for Dendrobium officinale polysaccharides.

[0006] The above-mentioned objective of this invention is achieved through the following technical solution:

[0007] This invention provides the application of Dendrobium officinale polysaccharide in the preparation of PGC-1α signaling pathway inhibitors.

[0008] This invention demonstrates that *Dendrobium officinale* can regulate the PGC-1α signaling pathway and the expression of metabolic genes in macrophages within the tumor microenvironment. It promotes macrophage M1 polarization, effectively inhibiting tumor growth and reducing tumor size. Simultaneously, it promotes glycolytic metabolism in tumor-associated macrophages, significantly enhancing the expression of macrophage glycolysis-related genes (Pgma1, Pkm, and Eno1) while inhibiting the expression of oxidative phosphorylation genes Nudfa5a, Dlat, and the PGC-1α gene, thus enhancing anti-tumor effects. Furthermore, *Dendrobium officinale* polysaccharides, by inhibiting the PGC-1α signaling pathway, can effectively regulate the body's anti-tumor immunity, suppress PGC-1α gene expression, and inhibit tumor occurrence and development, thus possessing a therapeutic effect on tumors. This invention provides an inhibitor that effectively regulates the PGC-1α pathway, and *Dendrobium officinale* polysaccharides, as an inhibitor of the PGC-1α signaling pathway, can be used to prepare anti-tumor drugs, providing a new approach for improving the metabolic state in the tumor microenvironment and for tumor immunotherapy.

[0009] Furthermore, the inhibitor can suppress PGC-1α gene expression.

[0010] Furthermore, the inhibitor enhances the anti-tumor effect by inhibiting the PGC-1α signaling pathway.

[0011] This invention provides the application of Dendrobium officinale polysaccharide as an inhibitor of the PGC-1α signaling pathway in the preparation of antitumor drugs.

[0012] This invention provides the application of Dendrobium officinale polysaccharide in the preparation of products that promote M1 macrophage polarization.

[0013] Furthermore, the product can promote the expression of IL-1β, IL-6 and NOS-2 in M1 macrophages.

[0014] Furthermore, the product can inhibit the expression of CD206 and ARG1 in M2 macrophages.

[0015] This invention provides the application of Dendrobium officinale polysaccharide in the preparation of reagents that enhance macrophage glycolytic metabolism.

[0016] Furthermore, the reagent can promote the expression of macrophage glycogenolysis-related genes Pgma1, Pkm, and Eno1.

[0017] Furthermore, the reagent can downregulate the expression of macrophage oxidative phosphorylation genes Nudfa5a and Dlat.

[0018] Preferably, the reagent can enhance the anti-tumor effect.

[0019] The present invention has the following beneficial effects:

[0020] This invention provides a novel application of Dendrobium officinale polysaccharide in the preparation of PGC-1α signaling pathway inhibitors. Studies have shown that Dendrobium officinale can regulate the PGC-1α signaling pathway and the expression of metabolic genes in macrophages within the tumor microenvironment. It can inhibit PGC-1α gene expression, promote macrophage M1 polarization, effectively inhibit tumor growth, and reduce tumor size. Simultaneously, it can promote glycolytic metabolism in tumor-associated macrophages, significantly promote the expression of macrophage glycolysis-related genes (Pgma1, Pkm, and Eno1), and inhibit the expression of oxidative phosphorylation genes Nudfa5a, Dlat, and PGC-1α genes, thereby enhancing the anti-tumor effect. Furthermore, Dendrobium officinale polysaccharides can effectively regulate the body's anti-tumor immunity by inhibiting the expression of the PGC-1α gene, promote macrophage M1 polarization, inhibit the occurrence and development of tumors, and have a therapeutic effect on tumors. It can also enhance the PGC-1α signaling pathway in macrophages. Therefore, Dendrobium officinale polysaccharides can be used to regulate the PGC-1α signaling pathway. As an inhibitor of the PGC-1α signaling pathway, it can be used to prepare anti-tumor drugs, providing a new approach for improving the metabolic state in the tumor microenvironment and for tumor immunotherapy. Attached Figure Description

[0021] Figure 1 The results of the effects of Dendrobium officinale polysaccharide on tumor-associated macrophage polarization genes are shown in the figure ((A) IL-1β level; (B) IL-6 level; (C) NOS-2 level; (D) CD206 level; (E) ARG1 level; Note: Compared with the control group, *P<0.05, **P<0.01).

[0022] Figure 2 The results of the effects of Dendrobium officinale polysaccharide on tumor-associated macrophage metabolism are shown in the figure ((A) expression of Pgma1 gene; (B) expression of Pkm gene; (C) expression of Eno1 gene; (D) expression of Nudfa5a gene; (E) expression of Dlat gene; Note: Compared with the control group, *P<0.05, **P<0.01).

[0023] Figure 3 The results of the effect of Dendrobium officinale polysaccharide on the PGC-1α gene in tumor-associated macrophages are shown in the figure ((A) expression of PGC-1α gene; (B) tumor volume; (C) correlation between tumor volume and PGC-1α gene; Note: compared with the control group, *P<0.05, **P<0.01). Detailed Implementation

[0024] The present invention will be further described below in conjunction with the accompanying drawings of the specification and specific embodiments, but the embodiments do not impose any form of limitation on the present invention. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the technical field.

[0025] Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

[0026] Example 1 Construction of a homologous tumor transplantation model of mouse colorectal cancer

[0027] Experimental animals: Male BALB / c mice (SPF grade, 6 - 8 weeks old, 18 - 22 g), purchased from the Experimental Animal Center of Guangzhou University of Chinese Medicine, license number: SYXK(Guangdong)2018 - 0085. The animals were housed in the SPF - level experimental animal room of the Experimental Animal Center of Guangzhou University of Chinese Medicine (20 - 25 °C, 65 - 70%), with a 12 - h light - dark cycle. During feeding, the animals were given standard feed and free access to sterile distilled water, and the bedding was changed every other day.

[0028] CT26 mouse colon cancer cells were purchased from iCell Bioscience Inc (Shanghai) and cultured in RPMI - 1640 medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 100 mg / mL streptomycin. The cells were cultured in a humidified incubator at 37 °C under specific conditions of 5% CO2. The polysaccharide from Dendrobium officinale was provided by Teacher Huang Song of the research group, and polysaccharide purification and structure identification were carried out.

[0029] According to the body weight of the mice, the mice were randomly divided into 5 groups, namely the control group, the 5 - FU positive drug group (using the anti - tumor drug 5 - fluorouracil, 50 mg / kg), and the low, medium, and high dose groups of polysaccharide from Dendrobium officinale (50, 100, and 200 mg / kg). 2×10 6 CT26 cells were suspended in 100 μL of 1640 medium and subcutaneously injected into the right forelimb of the mice. The tumor volume and body weight of the mice were measured twice a week, and the tumor volume was calculated. When the tumor volume was greater than 200 mm 3 ³, drug administration was started. The control group was intragastrically administered an equal volume of normal saline, the 5 - FU positive drug group was intraperitoneally injected with fluorouracil, and the polysaccharide from Dendrobium officinale was administered by gavage at the corresponding doses. At the end of the experiment, the mice were anesthetized and sacrificed, the tumors were removed, and the tumor weights were recorded.

[0030] Tumor macrophage extraction: Tumor tissue was minced and vortexed at 37°C for 75 min with 280 U / mL type 3 collagenase (Worthington Biochemical) and 4 μg / mL DNase I (Sigma). The mixture was then passed through a 70 μm sieve and separated using 44% and 66% Percoll gradients (Sigma). Cells were centrifuged at 3000 rpm for 30 min without immobilization. Cells at the 44% and 66% Percoll interfaces were collected and analyzed by flow cytometry.

[0031] Example 2: Expression of key polarization factors of macrophages M1 and M2 in tumor tissue

[0032] Macrophages were extracted from tumor tissues, and the expression of key genes M1 and M2 in macrophages was detected using an ELISA kit. Serum samples were collected from each group of animals, and the levels of macrophage polarization-related factors (IL-1β (Lianke Biotechnology, EK201B), IL-6 (Lianke Biotechnology, EK206), NOS-2 (ELISA, 17851), CD206 (ELISA, MM-1204M1), and ARG1 (Bioroyee, RE4163-96T)) in the serum were detected according to the ELISA kit instructions.

[0033] The measurement results are as follows Figure 1 As shown, Dendrobium officinale polysaccharide significantly promoted the expression of key factors (IL-1β, IL-6, NOS-2) in M1 macrophages (P<0.05 or P<0.01); while it significantly inhibited the expression of key factors (CD206, ARG1) in M2 macrophages (P<0.05 or P<0.01), indicating that Dendrobium officinale polysaccharide can promote M1 macrophage polarization in the tumor microenvironment and inhibit tumor growth.

[0034] Example 3: Expression of key metabolic genes in macrophages in tumor tissue.

[0035] Total RNA was isolated from tumor tissue using an RNA extraction kit (Aikerui Biotechnology, AG21101), and the concentration of total RNA was determined using a spectrophotometer. Then, following the instructions of a reverse transcription kit (Aikerui Biotechnology, AG11705), 1 μg of total RNA was reverse transcribed into cDNA. The expression of key macrophage metabolic genes Pgma1, Pkm, Eno1, Nudfa5a, and Dlat was detected using a SYBR Green kit (Aikerui Biotechnology, AG11701) and a CFX96 (Bio-Rad) amplification instrument. β-actin / GAPDH was used as an internal control. -△△Ct The value is used to calculate the relative expression level of the target gene mRNA.

[0036] Test results as follows Figure 2 As shown, after treating a homologous tumor transplantation model with Dendrobium officinale polysaccharide, Dendrobium officinale polysaccharide significantly promoted the expression of macrophage glycolysis-related genes (Pgma1, Pkm, and Eno1) (P<0.05 or P<0.01); at the same time, it significantly downregulated the expression of macrophage oxidative phosphorylation genes Nudfa5a and Dlat (P<0.05 or P<0.01), indicating that Dendrobium officinale polysaccharide can promote glycolytic metabolism in tumor-associated macrophages and promote macrophage M1 polarization.

[0037] Example 4: Expression of the PGC-1α pathway in macrophages of tumor tissue

[0038] The detection method in this embodiment is the same as in Example 3, using a CFX96 (Bio-Rad) amplification detector to detect the expression of the PGC-1α gene in macrophages. β-actin / GAPDH was used as an internal control, and the expression was analyzed using a 2... -△△Ct The relative expression level of the target gene mRNA was calculated, and the correlation between the expression of the PGC-1α gene and tumor volume was analyzed.

[0039] The results are as follows Figure 3 The results showed that Dendrobium officinale polysaccharide could dose-dependently inhibit the expression of the PGC-1α gene in tumor-associated macrophages; the high-dose group showed the most significant inhibitory effect on the PGC-1α gene, with statistically significant differences compared to the control group (P<0.01). Furthermore, after treatment with Dendrobium officinale polysaccharide in a homologous tumor transplantation model, the tumor volume was significantly reduced, with significant differences between the medium- and high-dose groups compared to the control group (P<0.05 or P<0.01). In addition, statistical analysis showed a significant positive correlation between PGC-1α gene expression and tumor size.

[0040] In summary, the results show that Dendrobium officinale polysaccharides can regulate the expression of metabolic genes in macrophages within the tumor microenvironment, inhibit the PGC-1α signaling pathway, promote macrophage M1 polarization, effectively inhibit tumor growth, and reduce tumor size. Simultaneously, it promotes glycolytic metabolism in tumor-associated macrophages, significantly enhances the expression of macrophage glycolysis-related genes (Pgma1, Pkm, and Eno1), and inhibits the expression of oxidative phosphorylation genes Nudfa5a, Dlat, and PGC-1α genes, thereby enhancing the anti-tumor effect and effectively reducing tumor volume and size. Furthermore, by inhibiting PGC-1α gene expression, Dendrobium officinale polysaccharides can effectively regulate the body's anti-tumor immunity, promote macrophage M1 polarization, and inhibit tumor occurrence and development, thus possessing a therapeutic effect on tumors. Therefore, this invention provides an inhibitor that can effectively regulate the PGC-1α pathway, and Dendrobium officinale polysaccharides, as an inhibitor of the PGC-1α signaling pathway, can be used to prepare anti-tumor drugs, providing a new approach for improving the metabolic state in the tumor microenvironment and for tumor immunotherapy.

[0041] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Application of Dendrobium officinale polysaccharide in the preparation of PGC-1α signaling pathway inhibitors.

2. The application according to claim 1, characterized in that, The inhibitor can suppress the expression of the PGC-1α gene.

3. The application according to claim 1, characterized in that, The inhibitor can enhance the anti-tumor effect.

4. Application of Dendrobium officinale polysaccharide as an inhibitor of the PGC-1α signaling pathway in the preparation of antitumor drugs.

5. Application of Dendrobium officinale polysaccharide in the preparation of products that promote the polarization of M1 macrophages.

6. The application according to claim 5, characterized in that, The product can promote the expression of IL-1β, IL-6 and NOS-2 in M1 macrophages.

7. The application according to claim 5, characterized in that, The product can inhibit the expression of CD206 and ARG1 in M2 macrophages.

8. Application of Dendrobium officinale polysaccharide in the preparation of reagents that enhance macrophage glycolysis metabolism.

9. The application according to claim 8, characterized in that, The reagent can promote the expression of macrophage glycogen-related genes Pgma1, Pkm, and Eno1.

10. The application according to claim 8, characterized in that, The reagent can downregulate the expression of macrophage oxidative phosphorylation genes Nudfa5a and Dlat.

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