Use of sodium butyrate in promoting proliferation of neural stem cells

Abstract

The application discloses a use of sodium butyrate in promoting proliferation of neural stem cells. The application finds that sodium butyrate has the effect of promoting proliferation of neural stem cells in vitro, which expands the functions of sodium butyrate as an inhibitor of histone deacetylase and the like. The application finds that adding sodium butyrate in an in-vitro culture medium can significantly enhance the proliferation ability of neural stem cells and effectively promote autophagy activity of the neural stem cells. The use can be used for stimulating regeneration of neural stem cells in vivo after injury or degeneration of the nervous system, thereby enhancing the repair ability, and can be used for improving the expansion ability of the neural stem cells cultured in vitro, thereby providing a high-quality cell source for transplantation treatment of nervous system diseases in vivo.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of drug research and development, and particularly relates to the use of sodium butyrate in promoting the proliferation of neural stem cells. BACKGROUND

[0002] Neural stem cells (NSCs) refer to cells present in the nervous system, which have the potential of self-renewal, proliferation and differentiation into various neural cells. After neurodegenerative diseases or nerve injury, the diseased site releases chemotactic factors to attract neural stem cells to migrate to the injury site and differentiate into different types of cells under the action of the local microenvironment to repair and supplement the damaged neural cells. However, with age, the number and proliferation capacity of neural stem cells in adults gradually decrease, and it is difficult to re-activate from the resting state to function, and the proliferation capacity of NSCs cultured in vitro is limited. Therefore, it is of great significance to extract stem cells and perform directional expansion in vitro, and then transplant them into the brain or spinal cord of patients with diseases such as nervous system injury and brain tumor to repair and reconstruct the brain nerves. How to effectively improve the proliferation capacity of NSCs in vitro and stimulate the regeneration capacity of neural stem cells in vivo after the nervous system is damaged or degenerative diseases occur has been a difficult problem that has not been solved.

[0003] Sodium butyrate (NaBu) has a molecular formula of C4H7O2Na, a molecular weight of 110.0869, a chemical name of Butanoic acid, sodium salt (1:1), and a CAS number of 156-54-7.

[0004] Its structural formula is as shown below:

[0005]

[0006] NaBu is also known as sodium n-butyrate, which is white to off-white in pure form, has a special cheese rancid smell, is easily soluble in water, and the aqueous solution has an alkaline pH. In vivo, NaBu is mainly generated by the fermentation of dietary fiber by butyric acid-producing bacteria in the colon, and is mainly used for energy supply by intestinal epithelial cells. NaBu promotes the entry of the M2 subtype of pyruvate kinase into the nucleus, up-regulates the expression of B-cell lymphoma-2 (Bcl-2), and then inhibits the apoptosis of myocardial cells. At the same time, NaBu is also an inhibitor of histone deacetylase (HDAC), which promotes the dissociation of DNA and histone, and then makes the transcription factor combine with DNA. NaBu can block the PI3K / AKT pathway to promote the apoptosis of various types of cancer cells. Moreover, NaBu can also play an antioxidant role by increasing the concentration of intracellular glutathione. At present, NaBu has been used in the research of intestinal anti-inflammatory and inhibition of cancer cell proliferation. However, whether NaBu can promote the proliferation of neural stem cells has not been reported. SUMMARY

[0007] The present application aims to provide the use of sodium butyrate in promoting the proliferation of neural stem cells.

[0008] The use of sodium butyrate in the preparation of a preparation for promoting the proliferation of neural stem cells.

[0009] Specifically, by adding sodium butyrate in the culture medium, the autophagy of in vitro isolated neural stem cells is promoted, and the proliferation of neural stem cells is promoted at the same time.

[0010] The use of sodium butyrate in the preparation of a drug for the proliferation of neural stem cells in vitro and in vivo.

[0011] A pharmaceutical composition for promoting the proliferation of neural stem cells in vitro and in vivo, comprising sodium butyrate and pharmaceutically acceptable salts thereof.

[0012] Preferably, it further comprises one or more pharmaceutically acceptable adjuvants or carriers.

[0013] The adjuvants or carriers include diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers or lubricants.

[0014] The pharmaceutical composition can be prepared into tablets, capsules, effervescent tablets, granules, powders, dispersible tablets, oral solutions, pills or injections.

[0015] The present application has the following beneficial effects: the present application finds that sodium butyrate has the effect of promoting the proliferation of neural stem cells, and sodium butyrate enhances the autophagy activity of neural stem cells at the same time. Sodium butyrate may promote the proliferation of neural stem cells by improving autophagy activity, and does not affect the stemness of neural stem cells. This effect can be used as an additive to prepare a drug and a culture medium for promoting the proliferation of neural stem cells, and is used to enhance the proliferation ability of neural stem cells in vitro and in vivo. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is the Western blot method for detecting the effects of different concentrations of butyrate on the proliferation and autophagy activity of neural stem cells cultured in vitro.

[0017] Figure 2 is the change in the size of neural spheres under a light microscope after treatment with different concentrations of butyrate. DETAILED DESCRIPTION

[0018] In order to facilitate the understanding of the present application, the present application will be described more fully below. However, the present application can be realized in many different forms, and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present application more thorough and comprehensive.

[0019] Example 1

[0020] I. Experimental materials

[0021] 1. DMEM / F12 medium, N2, B27, epidermal growth factor, basic fibroblast growth factor, Glutamax were purchased from Gibco, USA, sodium butyrate was purchased from Selleck, USA, product catalog number: S1999, CAS number 156-54-7, and used to prepare a 100 mM final concentration stock solution with pure water. After aliquoting, store at -20°C.

[0022] 2. Culture of neural stem cells

[0023] Take 13-day pregnant C57 pregnant mice, weigh and give 2% sodium pentobarbital anesthesia at 50 mg / kg. After sufficient anesthesia, prepare the skin, disinfect the surgical area with 75% alcohol, incise the skin to expose the pelvic cavity, and after disinfection of the uterus in 75% alcohol, place it in pre-cooled PBS, carefully separate the fetal mouse from the uterus and place it in another pre-cooled PBS culture dish, wash the residual blood, and separate the forebrain and midbrain of the fetal mouse under a dissecting microscope using ophthalmic forceps. After stripping the dura mater, the separated brain tissue was cut into a paste and gently blown on ice, and after single cell formation, it was screened with a 40 μm filter screen. After collecting the filtrate, centrifuge at 1200 rpm for 5 min. Discard the supernatant, add complete culture medium (DMEM / F12 + 2% B27 + 1% N2 + 0.2 μg / L epidermal growth factor + 0.2 μg / L basic fibroblast growth factor + 1% Glutamax), count the neural stem cells, adjust the cell density to 5 x 10 5 6 / mL, inoculate into a cell culture flask, and incubate in a 37°C, 5% CO2 incubator. Collect the neurospheres after 3-4 days of culture, centrifuge at 800 rpm for 5 min, discard the supernatant, digest with Accutase for 10 min, terminate the digestion with DMEM / F12 medium, centrifuge again, discard the supernatant, and add fresh complete culture medium, and record it as F1.

[0024] II. Experimental grouping

[0025] Take the third generation of neural stem cells and intervene as follows:

[0026] Control group: use complete culture medium;

[0027] Butyrate intervention group: add butyrate to the culture medium to a final concentration of 1 μM and 10 μM.

[0028] III. Experimental methods

[0029] 1. Western Blot experiment

[0030] Neural stem cells were treated according to their respective groups for 12 hours, then collected by centrifugation at 2,000 rpm for 5 minutes, and the supernatant was discarded. An appropriate amount of RIPA Lysis Buffer (with protease inhibitors added before use) was added, and the mixture was thoroughly mixed. The cells were placed on ice for 30 minutes to allow for complete lysis. After centrifugation at 12,000 rpm for 10 minutes, the supernatant was aspirated and thoroughly mixed with loading buffer. The mixture was then incubated at 100°C for 10 minutes for Western blotting analysis. Following electrophoresis, transfer to a membrane, and blocking, antibody incubation and development were performed to detect changes in the expression of proliferating cell nuclear antigen (PCNA) and autophagy marker molecule LC3 (microtubule-associated protein 1 light chain 3).

[0031] The antibodies used in the experiment are shown in the table below:

[0032]

[0033] 2. Observation and photography using a light microscope

[0034] The culture flasks were observed under a light microscope, and representative neurospheres were photographed.

[0035] Experimental results:

[0036] like Figure 1 As shown, after treatment with butyrate, neural stem cells showed that, compared with the control group, butyrate significantly promoted the expression of PCNA and LC3II, increased autophagy activity, and exhibited a concentration-dependent effect. Simultaneously, under a light microscope, it was observed that the neurospheres treated with butyrate were more plump and larger, indicating an increased number of neural stem cells. Figure 2 The above results indicate that sodium butyrate promotes the proliferation of neural stem cells and may regulate proliferation by affecting autophagy activity.

[0037] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Use of sodium butyrate for the preparation of a preparation for promoting the proliferation of neural stem cells, characterized in that, By adding sodium butyrate in the culture medium, the proliferation of the in vitro isolated neural stem cells is promoted, while the autophagy of the neural stem cells is promoted.

Images