Application of paris polyphylla saponin in preparation of preparation for inhibiting SREBP1 pathway mediated lipid metabolism

Abstract

The invention discloses application of paris polyphylla saponin in preparation of a preparation for inhibiting SREBP1 pathway activity, the preparation for inhibiting the SREBP1 pathway activity achieves the anti-tumor purpose by inhibiting SREBP1 pathway mediated lipid metabolism, and a series of experiments prove that the paris polyphylla saponin can significantly down-regulate the expression of SREBP1, so that the SREBP1 pathway activity can be inhibited, and the SREBP1 pathway activity can be inhibited. The invention provides a novel target spot and drug candidate for tumor treatment and has good clinical application prospect. The novel target spot and drug candidate can be used for inhibiting the tumor cell proliferation and inducing the tumor cell apoptosis by inhibiting the transcription of downstream lipid metabolism related genes and reducing the synthesis and accumulation of lipids in tumor cells, so that the novel target spot and drug candidate are provided for the tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of Paris polyphylla saponins in the preparation of a preparation for inhibiting lipid metabolism mediated by the SREBP1 pathway to exert an anti-tumor effect. Background Art

[0002] Cancer is one of the major diseases that seriously threatens human health. Its occurrence and development is a complex multi-step process involving the abnormal regulation of multiple genes and signaling pathways. Lipid metabolism reprogramming is one of the important characteristics of tumor cells. Tumor cells meet the energy and biosynthetic raw materials required for their rapid proliferation by enhancing lipid synthesis and uptake. SREBP1 (sterol regulatory element binding protein 1) is a key transcription factor that regulates lipid metabolism. It is often highly expressed in tumor cells. By activating the expression of a series of downstream lipid metabolism-related genes (such as FASN, SCD1, etc.), it promotes lipid synthesis and supports the growth and metastasis of tumor cells. Therefore, inhibiting lipid metabolism mediated by the SREBP1 pathway has become an important strategy for anti-tumor treatment. Paris polyphylla (Paridis yunnanensis) is a traditional Chinese medicinal herb with benefits such as clearing heat and detoxifying, reducing swelling and alleviating pain. Modern pharmacological studies have shown that Paris polyphylla possesses anti-tumor activity, with Paris polyphylla saponins as its main active ingredient. However, the specific molecular mechanisms underlying the anti-tumor effects of Paris polyphylla saponins remain unclear, particularly whether they exert their anti-tumor effects through regulation of lipid metabolism mediated by the SREBP1 pathway. Summary of the Invention

[0003] The purpose of the present invention is to provide a new use of Paris polyphylla saponins, namely, the use of Paris polyphylla saponins in the preparation of an active preparation for inhibiting the SREBP1 pathway, which achieves the purpose of anti-tumor by inhibiting lipid metabolism mediated by the SREBP1 pathway.

[0004] The Paris polyphylla saponin is prepared by extracting crushed Paris polyphylla 1-3 times at 30-70° C. under ultrasound using a cyclodextrin solution, collecting and combining the extracts, separating and purifying the extracts with a macroporous resin, eluting with a 70% volume concentration ethanol solution, and concentrating and drying the eluate.

[0005] Tumors include bladder cancer, liver cancer, lung cancer, breast cancer, and colon cancer.

[0006] The ingredients (or active ingredients) of the preparation of the present invention are Paris polyphylla saponins, and one or more pharmaceutically acceptable excipients may be added, or the preparation may be compounded with other active ingredients to exert an inhibitory effect; in addition to being made into tablets, the preparation may also be made into various pharmaceutically acceptable forms such as pills, powders, capsules, granules, oral liquids and injections.

[0007] Compared with the prior art, the present invention has the following advantages: This study, published in the journal Nature Communications, demonstrates that polysaccharides from Paridis yunnanensis can inhibit the SREBP1 pathway, downregulating the expression of downstream lipid metabolism genes, reducing lipid synthesis and accumulation in tumor cells, thereby inhibiting tumor cell proliferation and inducing apoptosis, exerting anti-tumor effects. This study provides a new theoretical basis for the clinical application of polysaccharides from Paridis yunnanensis, as well as new targets and drug candidates for tumor treatment, possessing significant theoretical and clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The results of the effect of PPT on the protein expression of the SREBP1 pathway in T24 cells are shown in the upper figure, which is a Western blot, and the lower figure is an expression statistical chart; Figure 2 The results of the effect of PPT on the protein expression of the SREBP1 pathway in UM-UC-3 cells are shown in the figure above, which is the protein electrophoresis diagram and the figure below is the expression level statistical diagram; Figure 3 The results show the effects of PPT and SC79 on the protein expression of the SREBP1 pathway in T24 cells. The upper figure is a Western blot, and the lower figure is an expression statistical graph. Figure 4 The results show the effects of PPT and SC79 on the protein expression of SREBP1 pathway in UM-UC-3 cells. The upper figure is the protein electrophoresis diagram, and the lower figure is the expression level statistical diagram; Figure 5 The results show the effects of PPT and POA on the protein expression of SREBP1 pathway in T24 cells. The upper figure is a Western blot, and the lower figure is an expression statistical graph. Figure 6 The results show the effects of PPT and POA on the protein expression of SREBP1 pathway in UM-UC-3 cells. The upper figure is a Western blot, and the lower figure is an expression level statistical graph. Figure 7 This is the result of the effects of PPT and POA on T24 cell death; Figure 8 This is the result of the effects of PPT and POA on UM-UC-3 cell death. DETAILED DESCRIPTION

[0009] The technical solutions of the present invention are further described below in conjunction with the accompanying drawings and specific embodiments. However, these embodiments should not be used to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are commercially available unless otherwise specified. After reading the description of the present invention, various equivalent changes, modifications, and modifications made by technicians in this field are within the scope defined by the claims of the present invention.

[0010] In the embodiment, Paris polyphylla ( Paris polyphylla Smith var. yunnanensis The dried rhizomes of Hand.-Mazz were purchased from the Kunming medicinal material market in Yunnan Province and were identified as authentic.

[0011] Reagents: RPMI 1640 medium (Gibco, USA), fetal bovine serum (Guangzhou Saiku Biotechnology Co., Ltd.), trypsin (Gibco, USA), hydroxypropyl- β -cyclodextrin (Aladdin, Shanghai, China), Opti-MEM medium (Gibco, USA), palmitoleic acid (MCE, Shanghai, China), SC79 (MCE, Shanghai, China).

[0012] Cell lines: Bladder cancer cell lines T24 and UM-UC-3 were purchased from Guangzhou Saiku Biotechnology Co., Ltd.

[0013] Example 1: Polygonum multiflorum saponins inhibit the SREBP1 pathway 1. Crush the dried rhizome of Paris polyphylla and pass it through a 200-mesh sieve to obtain coarse powder; 2. Add 2% hydroxypropyl- β -cyclodextrin solution, after mixing, ultrasonic extraction (power 180W) at 50 ° C for 30 minutes, solid-liquid separation, solid and then add hydroxypropyl- β -The cyclodextrin solution was extracted twice, and the combined extracts were collected and passed through a macroporous resin HPD600 column. The extracts were eluted with a 70% ethanol solution by volume. The eluate was concentrated at 40°C and freeze-dried to obtain Paris polyphylla saponins (PPT). 3. Western Blotting Bladder cancer cells T24 and UM-UC-3 in the logarithmic growth phase were evenly seeded into 6-well plates and placed in a cell culture incubator. The plates were cultured at 37°C and 5% CO2 for 24 hours. After the cells were completely attached, they were treated with different concentrations of PPT (7.5 μg / mL, 15 μg / mL, and 30 μg / mL) for 24 hours. Total cell protein was extracted and western blotting was performed to detect the expression levels of p-Akt, p-mTOR, p-P70S6K, SREBP1, ACC, FASN, SCD1, and GAPDH proteins. See the results Figure 1 、 2 ,It can be seen from the figure that PPT dose-dependently inhibited the expression of key proteins of the SREBP1 pathway, including SREBP1, ACC, FASN, SCD1, p-Akt, and p-P70S6K.

[0014] Example 2: SC79 attenuates the inhibitory effect of PPT on the SREBP1 pathway Bladder cancer cells T24 and UM-UC-3 in the logarithmic growth phase were evenly seeded into 6-well plates and placed in a cell culture incubator. They were cultured at 37°C and 5% CO2 for 24 hours. After the cells were completely attached, they were treated with 20 μg / mL PPT and / or 30 μM SC79 for 24 hours, and then the total cell protein was extracted. Western blotting was performed to detect the expression levels of p-Akt, p-mTOR, SREBP1, FASN, SCD1, C-PARP, and GAPDH proteins. A blank plate (without PPT or SC79) was also set up. See the results Figure 3 、 4 As can be seen from the figure, the Akt activator SC79 can effectively rescue PPT-inhibited cell apoptosis (evidenced by reduced c-PARP levels) and restore SREBP1 pathway protein expression.

[0015] Example 3: Palmitoleic acid (POA) attenuates PPT-inhibited cell apoptosis T24 and UM-UC-3 bladder cancer cells in the logarithmic growth phase were evenly seeded into 6-well plates and placed in a cell culture incubator. They were cultured at 37°C and 5% CO2 for 24 hours. After the cells were fully attached, they were treated with 20 μg / mL PPT and / or 100 μM palmitoleic acid (POA) for 24 hours. Total cell protein was extracted and western blotting was performed to detect the expression levels of C-PARP, C-cas-7, and GAPDH proteins. A blank plate (no PPT or POA) was also set up. See the results Figure 5 、 6 ,As can be seen from the figure, the Western blot results showed that the C-PARP and C-cas-7 protein levels decreased after the addition of POA, and ,significantly alleviated the cell apoptosis inhibited by PPT (p<0.05).

[0016] Example 4: POA alleviates PPT-inhibited cell death Bladder cancer cell lines T24 and UM-UC-3 were evenly plated into 96-well plates (4000 cells / well, 100 μL per well), placed in a cell culture incubator, and cultured at 37°C, 5% CO2 for 24 hours. After the cells were completely attached, they were treated with different concentrations (1.5625 μg / mL, 3.125 μg / mL, 6.25 μg / mL, 12.5 μg / mL, 25 μg / mL, 50 μg / mL) of Paris polyphylla saponins (PPT), different concentrations of PPT, and 100 μM POA for 48 hours. Then, 30 μL of MTT solution (5 mg / mL) was added to the 96-well plate, placed in an incubator, and reacted at 37°C, 5% CO2 for 5 hours. Then, the liquid in the well plate was discarded, and 100 μL of DMSO solution (purity 99.99%) was shaken on a shaker for 10 min (20 rpm). The absorbance was measured at 570 nm. The cell viability was calculated, the dose-effect curve was drawn, and the IC50 value was calculated. See the results Figure 7 、 8 From the figure, we can see that Paris polyphylla saponins can inhibit the proliferation of bladder cancer cells. After adding POA, it significantly reduced the death of bladder cancer cells induced by PPT.

[0017] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in the present invention should be included in the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. Use of a Paris polyphylla saponin in the preparation of an active preparation for inhibiting the SREBP1 pathway.

2. The use according to claim 1, characterized in that: Paris polyphylla saponins are prepared by extracting crushed Paris polyphylla 1-3 times with a cyclodextrin solution at 30-70°C under ultrasound, collecting and combining the extracts, separating and purifying the extracts with a macroporous resin, eluting with a 70% volume concentration ethanol solution, and concentrating and drying the eluate.

3. The use according to claim 1, characterized in that: Preparations that inhibit the activity of the SREBP1 pathway achieve anti-tumor effects by inhibiting lipid metabolism mediated by the SREBP1 pathway.

4. The use according to claim 3, characterized in that: Tumors include bladder cancer, liver cancer, lung cancer, breast cancer, and colon cancer.

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