Medicine for treating alcohol-induced bone marrow mesenchymal stem cell osteogenic differentiation disorder

Abstract

The invention relates to the field of medicines, in particular to a medicine for treating alcohol-induced stem cell osteogenic differentiation disorder and a preparation method and application thereof. The medicine is prepared from the following raw materials in parts by weight: 1 part of naringin and 0.33 to 7 parts of morroniside. The medicine is prepared into tablets, pills, powder, hard capsules, soft capsules, granules and oral liquid by adopting a conventional method in a medicinal preparation. The morroniside and naringin composition is applied to medicines for treating METTL14 gene silencing alcohol-induced bone marrow mesenchymal stem cell osteogenic differentiation disorder.

Description

Technical field:

[0001] The present invention relates to the field of medicines, and in particular to a pharmaceutical composition consisting of morroniside and naringin and a preparation method thereof. Technical background:

[0002] Morroniside is a terpenoid glycoside compound purified from the ripe pulp of Cornus officinalis or Elderberry. The purified morroniside is a white crystalline powder with a molecular formula of C 17 H 26 O 11 , with a relative molecular mass of 406.3817 (molecular structure shown in Formula I). ​​As a natural small molecule monomer compound, morroniside has been shown to have multiple biological activities such as anti-inflammatory, anti-coagulation, anti-apoptosis, anti-oxidative stress and promoting repair.

[0003]

[0004] Naringin is a flavonoid-flavanone aglycone component found in common herbal medicines such as Citrus aurantium, Citrus aurantium, and Rhizoma Drynariae. Its chemical formula is C 27 H 32 O 14， The relative molecular mass is 580.53 (molecular structure is shown in Formula II). Studies have shown that naringin has certain effects in anti-inflammation, anti-oxidation, regulating cell cycle, hormone metabolism, and protecting bone tissue.

[0005]

[0006] In 2019, Shen Yiwei, Li Xue and others published a paper titled "Research Progress on Naringin in Drynaria for the Treatment of Femoral Head Necrosis" in the Journal of Liaoning University of Traditional Chinese Medicine (Shen Yiwei, Xu Xilin, Zhang Xiaofeng, Li Xue, Xie Xinsheng, Yu Changsui, Lv Hang, Fang Xiangchun, He Xuefeng, Li Xiaodong. Research Progress on Naringin in Drynaria for the Treatment of Femoral Head Necrosis [J]. Journal of Liaoning University of Traditional Chinese Medicine, 2019, 21(07):128-131.). The paper proposed that naringin (NG) is the main active monomer component of the traditional Chinese medicine Drynaria, a dihydrotestosterone flavonoid compound, which has the functions of healing fractures, strengthening tendons and bones, warming kidney yang, and regulating bone metabolism. Femoral head necrosis is a multifactorial bone disease with various treatment methods. Early treatment aims to delay the course of the disease, prevent and treat collapse, and maximize the preservation of hip function. Naringin can be used to treat femoral head necrosis. It can promote osteoblast proliferation and differentiation to benefit bone formation, inhibit osteoclast absorption of bone, inhibit adipogenic differentiation and inflammatory response, inhibit endothelial cell apoptosis, promote endothelial progenitor cell proliferation and angiogenesis, increase the length and area of ​​vascular formation, facilitate bone necrosis repair, and reduce the rate of femoral head necrosis.

[0007] In addition, Shen Yiwei, Li Xue, et al. published a paper titled "Effects of naringin on alcohol-induced oxidative stress, autophagy, apoptosis and osteogenic adipogenic differentiation of bone marrow mesenchymal stem cells" in the journal "Chinese Journal of Traditional Chinese Medicine" in 2022 (Shen Yiwei, Li Xue, Zhang Xiaofeng, Xu Xilin, Li Xiaodong, Hou Bin, Lu Hang, Li Zuo. Effects of naringin on alcohol-induced oxidative stress, autophagy, apoptosis and osteogenic adipogenic differentiation of bone marrow mesenchymal stem cells [J]. Chinese Journal of Traditional Chinese Medicine, 2022, 40(05): 36-39+263-265.), which systematically explored the effects of naringin on alcohol-induced oxidative stress, autophagy, apoptosis and osteogenic adipogenic differentiation of bone marrow mesenchymal stem cells. Bone marrow mesenchymal stem cells were purchased from the ATCC cell bank in the United States. The blank group, alcohol group and traditional Chinese medicine group were randomly set up. The blank group was given basal culture medium or induction culture medium, the alcohol group was given 100mmoL / L alcohol on the basis of the blank group, and the traditional Chinese medicine group was given 100mmoL / L naringin on the basis of the alcohol group. The MTS method was used to detect cell viability, the H2DCFDA method was used to mark the intracellular reactive oxygen content, the Western Blot method was used to detect the expression level of the autophagy key gene LC3 protein, and the flow cytometry method was used to detect cell apoptosis. Alizarin red staining was used to evaluate the effects of alcohol and naringin on the formation of BMSCc mineralized nodules, and Oil Red O staining was used to evaluate the effects of alcohol and naringin on the formation of lipid droplets during adipogenic differentiation of BMSCc. The results showed that compared with the blank group and the alcohol group, naringin could antagonize the increase in reactive oxygen content induced by alcohol, reduce the expression of LC3 protein and apoptosis level, inhibit lipid formation and promote the formation of mineralized nodules. Naringin can antagonize the increase in oxidative stress induced by alcohol, regulate cell autophagy, inhibit apoptosis, and thus restore the balance of osteogenic / adipogenic differentiation of BMSCs.

[0008] Alcohol inhibits the osteogenic differentiation of stem cells, leading to osteogenic differentiation disorder, which is the core pathogenesis of alcoholic bone disease and the key to preventing and treating the disease. qRT-PCR (Quantitative real-time polymerase chain reaction analysis) and Alizarin red staining confirmed that alcohol (100mmoL / L) inhibited the expression of stem cell osteogenic differentiation marker genes (Runx2, ALP, BMP2, SP7, etc.) and the formation of calcified nodules, leading to osteogenic differentiation disorder.

[0009] m6A modification can be used as a drug target. m6A (N6-methyladenosine) modification is a new epitranscriptome RNA modification. m6A modification is reversible and dynamically regulated, and plays a vital role in a variety of biological processes and diseases. The m6A methyltransferase complex is known to contain methyltransferases ETTL3 and METTL14, the METTL3 adaptor WTAP4, and other related proteins, including KIAA1429, RBM15 / 15B, and ZC3H13. In the METTL3-METTL14 methyltransferase domain complex, METTL3 acts as a catalytic subunit, while METTL14 provides an RNA binding scaffold to activate and enhance the catalytic activity of METTL3. WTAP has no catalytic activity, but can interact with METTL3 and METTL14 to regulate the m6A level of RNA transcription. METTL3 and METTL14 play important roles in different biological processes such as embryonic development and neurogenesis. It has been confirmed that METTL3 can also regulate cell reprogramming, spermatogenesis, T cell homeostasis and endothelial-hematopoietic transformation. METTL14 is reported to be a tumor suppressor gene in glioblastoma and plays an oncogenic role in the development and maintenance of acute myeloid leukemia. Increasing evidence supports that m6A modification may play an important role in regulating gene expression, splicing, RNA editing, RNA stability, controlling mRNA lifespan and degradation, and regulating gene expression to regulate bone development. Post-transcriptional m6A modification and its related regulatory factors have important biological functions in bone development and bone remodeling. Studies have shown that m6A methyltransferase METTL14 can promote bone formation by upregulating TCF1 gene expression through m6A mRNA methylation, thereby playing an anti-osteoporotic role. METTL14 promotes osteoblast differentiation and inhibits osteoclast activation by upregulating SIRT1 mRNA levels in an m6A-dependent manner, thereby alleviating the development of osteoporosis. m6A methyltransferase METTL3 and demethyltransferase FTO are involved in the delicate process between adipogenic differentiation and osteogenic differentiation of BMSCs. Demethylase FTO regulates the differentiation fate of BMSCs through GDF11-FTO-PPARγ signal transduction, promotes the transformation of BMSCs into adipocytes and inhibits bone formation. At the cellular level, m6A modification affects the proliferation, differentiation and apoptosis of BMSCs, osteoblasts and osteoclasts by regulating the expression of osteogenic marker genes (Runx2, ALP, Osterix). In addition, signaling pathways such as PTH / Pth1r, PI3K-Akt, Wnt / β-Catenin, which play an important role in bone remodeling regulation, are also regulated by m6A. It can be inferred that targeting Runx2 post-transcriptional m6A modification can be used as a new potential therapeutic target for regulating bone metabolic balance, and it is worth exploring and studying the development of drugs to prevent and treat alcohol-induced osteogenic differentiation disorders of stem cells.

[0010] No feast is complete without wine. The drinking culture is prevalent. Long-term drinking can increase the risk of alcoholic osteonecrosis, alcoholic osteoporosis, etc. A study by the international top journal "The Lancet" shows that alcoholism causes many damages to the body and is the seventh largest risk factor for death in the world. Globally, among people aged 15 to 49, about 3.8% of female mortality and about 12.2% of male mortality can be attributed to alcoholism. In Asia, femoral head necrosis caused by long-term heavy drinking accounts for 32.4% to 45.3% of non-traumatic femoral head necrosis. With the improvement of living standards and the sharp increase in the number of drinkers, the incidence of alcoholic osteoporosis, alcoholic femoral head necrosis, etc. has increased year by year. Due to the lack of recognized and effective prevention and treatment methods, alcoholic bone disease has become one of the medical problems that need to be solved urgently. Alcoholic bone disease belongs to the categories of "alcoholic disease", "drunkenness", "bone atrophy", "bone withering", "bone injury", "bone brittleness", "bone extreme" and "bone sparsity". The cause of the disease is alcohol poisoning, and the disease is located in the tendons and bones. Traditional Chinese medicine can play a positive role in intervening in the occurrence and development of alcoholic bone remodeling, and it is a treasure trove that needs to be explored. Alcoholic bone remodeling is characterized by abnormal bone quality and bone mass throughout the body. It is a comprehensive reflection of bone homeostasis imbalances such as osteoblastic / osteoclast imbalance, osteoblastic / adipogenic imbalance, metabolic disorders, stress instability, and aging degeneration induced by long-term alcohol intake. It is the core pathogenesis of bone diseases such as alcoholic osteonecrosis and alcoholic osteoporosis. Alcoholic bone disease represented by alcoholic femoral head necrosis and alcoholic osteoporosis is common in clinical practice, and there is no effective treatment plan; alcoholic bone remodeling is its core pathogenesis. It is an effective way to overcome alcoholic bone disease to explore ways to intervene in alcoholic bone remodeling from the perspective of traditional Chinese medicine and give full play to the unique advantages of traditional Chinese medicine in multi-component, multi-target, and multi-dimensional coordinated integration.

[0011] Rat bone marrow mesenchymal stem cells (rBMSCs) can differentiate into a variety of mesodermal tissue cells (such as bone, cartilage, muscle, fat, etc.) and other germ layer cells (such as neurons). Studies have confirmed that bone differentiation disorders of bone marrow mesenchymal stem cells play a key role in the course of alcoholic bone disease (alcoholic osteoporosis, alcoholic osteonecrosis, etc.), and regulating the osteogenic differentiation of stem cells is also the key to preventing and treating alcoholic bone disease.

[0012] In addition, Huang Haozhe, Zheng Min, Li Na and other researchers published a paper titled "Effects of naringin on bone healing of tooth extraction wounds in osteoporotic rats" in the journal "Chinese Journal of Tissue Engineering Research" in 2023; Zhou Zheren, Ma Xun, Mo Huankai and other researchers published a paper titled "Effects of combined use of naringin and luteolin on the expression of genes related to the Wnt / β-cantein pathway during rat BMSCs-induced osteogenesis" in the journal "Advances in Modern Biomedicine" in 2023; Li Shengting, Yao Yizhang, Zhao Guoting and other researchers published a paper titled "Naringin regulates lncRNA MEG3 / Wnt / β-catenin signaling pathway promotes osteogenic differentiation of inflammatory-derived periodontal ligament stem cells"; Zhang Liangliang, Zhao Chengjin, Zhou Yuhu and other researchers published a paper in the journal Hebei Medicine in 2022 titled "Naringin promotes osteogenic differentiation of human adipose-derived mesenchymal stem cells through cAMP / PKA / CREB pathway". These research results have more or less confirmed that naringin can promote osteogenic differentiation of stem cells. However, these studies did not involve the medicinal value of naringin in the treatment of alcohol-induced stem cell osteogenic differentiation disorders.

[0013] Yuan Xu, Dong Peiliang, Han Hua and other researchers published a paper in the journal "Central South Pharmacy" in 2022, "Study on the activity of elderberry active ingredient - morroniside on osteoblasts" Yang Houzhi, Dong Runbei, Jia Yutao, Li Yuqiao, Luo Gan, Li Tianhao...Sun Tianwei (2023), Morroniside improves glucocorticoid-induced osteoporosis and promotes osteoblast formation by interacting with sodium-glucose co-transporter 2. Biopharmaceuticals (1), 416-426; Li Xi, Zhu Yunrong, Lin Xiangquan, Chen Chuanyuan, Liu Hui, Shi Yi (2022), Beclin1 and atg13-dependent autophagy activation has a synergistic effect with morroniside on osteoblasts, Experimental Biology and Medicine (Maywood, New Jersey) (2019), 15353702221116879-15353702221116879. Dong Runbei, Jia Yutao, Yang Houzhi, Luo Gan, Li Yuqiao, Sun Tianwei, (2022), Effects and mechanisms of morroniside on osteogenic differentiation and proliferation of mouse MC3T3-E1 cells. Chinese Journal of Reparative and Reconstructive Surgery (7), 889-895. Liu Hui, Li Xi, Lin Jingui, Lin Miaoguo. (2021), Morroniside promotes osteogenesis by activating PI3K / Akt / mTOR signaling, Bioscience and Technology (2), 332-339. Sun Yi, Zhu Yu, Liu Xuanzhe, … Xu Jia (2020). Morroniside alleviates high glucose-induced BMSC dysfunction by regulating the Glo1 / AGE / RAGE axis, Cell Proliferation (8), e12866. Hu Naiwu, Ren Shujun, Li Wei, … Zhao Chengbin, (2013), Morroniside promotes proliferation of rat bone marrow mesenchymal stem cells, Molecular Medicine Reports (5), 1565-70. These research results have more or less confirmed that morroniside can promote the osteogenic differentiation of stem cells. However, none of these studies have involved the medicinal value of morroniside in the treatment of alcohol-induced osteogenic differentiation disorders of stem cells. Summary of the invention:

[0014] The purpose of the present invention is to provide a medicine of naringin and morroniside.

[0015] Another object of the present invention is to provide a method for preparing the drug.

[0016] The invention also provides pharmaceutical use of the medicine.

[0017] The object of the present invention is achieved by the following methods:

[0018] A medicine is prepared from the following raw materials in parts by weight: naringin and morroniside.

[0019] The drug is a drug for treating alcohol-induced osteogenic differentiation disorder of bone marrow mesenchymal stem cells in which the METTL14 gene is not silenced, and is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 0.33-7 parts by weight of morroniside.

[0020] As a preferred technical solution of the above-mentioned medicine, the medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 2 parts by weight of morroniside.

[0021] As a preferred technical solution of the above-mentioned medicine, the medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 3 parts by weight of morroniside.

[0022] The above-mentioned medicine made from naringin and morroniside as raw materials is a medicine for treating alcohol-induced osteogenic differentiation disorder of bone marrow mesenchymal stem cells with METTL14 gene silencing. The medicine is made from the following raw materials in parts by weight: 1 part by weight of naringin and 2-4 parts by weight of morroniside.

[0023] As a preferred technical solution of the above-mentioned medicine, the medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 2 parts by weight of morroniside.

[0024] As a preferred technical solution of the above-mentioned medicine, the medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 3 parts by weight of morroniside.

[0025] As a preferred technical solution of the above-mentioned medicine, the medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 4 parts by weight of morroniside.

[0026] The above-mentioned drugs can be prepared into tablets, pills, powders, hard capsules, soft capsules, granules and oral liquids by conventional methods in pharmaceutical preparation.

[0027] The invention discloses an application of 1 weight part of naringin and 2-4 weight parts of morroniside as raw materials in preparing a medicine for treating alcohol-induced osteogenic differentiation disorder of bone marrow mesenchymal stem cells with METTL14 gene silencing.

[0028] Naringin is isolated and extracted from Drynaria fortunei, and morroniside is isolated and extracted from Cornus officinalis and Elderberry. The purity is ≥95% (HPLC) and can also be purchased directly in the market.

[0029] The invention discloses an application of 1 weight part of naringin and 2-4 weight parts of morroniside as raw materials in preparing a medicine for treating alcohol-induced osteogenic differentiation disorder of bone marrow mesenchymal stem cells with METTL14 gene silencing. Description of the drawings:

[0030] Figure 1 :Western Blot was used to detect the protein expression of m6A modification-related gene METTL14;

[0031] Figure 2: Positive control group (0.92±0.07), after staining with Alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0032] Figure 3 : In the blank control group (0.09±0.05), after staining with Alizarin red, no calcium salt deposition in the cell mineralized nodules appeared orange-red, and no significant mineralized nodules were formed;

[0033] Figure 4 : Model group (0.39±0.19), after staining with Alizarin red, the calcium salt deposition in the mineralized nodules of cells appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0034] Figure 5 : Experimental drug group A: Naringin (0.41±0.21), after staining with Alizarin Red, the calcium salt deposition in the mineralized nodules of cells appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0035] Figure 6 : Experimental drug group B: Morroniside (0.40±0.22), after staining with Alizarin Red, the calcium salt deposition in the mineralized nodules of cells appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0036] Figure 7 : Experimental drug group C: naringin: morroniside = 3:1 (0.38±0.19), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0037] Figure 8 : Experimental drug group D: naringin: morroniside = 2:1 (0.39±0.21), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0038] Fig. 9 : Experimental drug group E: naringin: morroniside = 1:1 (0.37±0.22), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0039] Fig.10 : Experimental drug group F: naringin: morroniside = 1:2 (0.79±0.22), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0040] Fig.11 : Experimental drug group G: naringin: morroniside = 1:3 (0.89±0.19), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0041] Fig.12 : Experimental drug group H: naringin: morroniside = 1:4 (0.71±0.23), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0042] Fig.13 : Experimental drug group I: naringin: morroniside = 1:5 (0.41 ± 0.20), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0043] Fig.14 : Experimental drug group J: naringin: morroniside = 1:6 (0.39 ± 0.19), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0044] Fig.15 : Experimental drug group K: naringin: morroniside = 1:7 (0.41±0.24), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0045] Fig.16 : Control-model group: (0.56±0.24), after staining with Alizarin red, the calcium salt deposition in the mineralized nodules of cells appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0046] Fig.17 : Control-experimental drug group A: Naringin (0.79±0.19), after staining with Alizarin Red, the calcium salt deposition of the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0047] Fig.18 : Control-experimental drug group B: Morroniside (0.83±0.18), after staining with Alizarin Red, the calcium salt deposition in the mineralized nodules of cells appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0048] Fig.19: Control-experimental drug group C: naringin: morroniside = 3:1 (0.69±0.17), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0049] Fig. 20 : Control-experimental drug group D: naringin: morroniside = 2:1 (0.89±0.20), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0050] Fig.21 : Control-experimental drug group E: naringin: morroniside = 1:1 (0.86±0.21), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0051] Fig. 22 : Control-experimental drug F group: naringin: morroniside = 1:2 (0.87±0.19), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows in the figure;

[0052] Fig.23 : Control-experimental drug G group: naringin: morroniside = 1:3 (0.86±0.21), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0053] Fig.24 : Control-experimental drug H group: naringin: morroniside = 1:4 (0.90±0.26), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0054] Fig.25 : Control-experimental drug group I: naringin: morroniside = 1:5 (0.89±0.25), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0055] Fig.26 : Control-experimental drug group J: naringin: morroniside = 1:6 (0.889 ± 0.19), after staining with alizarin red, the calcium salt deposition of the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown as black arrows in the figure;

[0056] Fig. 27: Control-experimental drug group K: naringin: morroniside = 1:7 (0.89±0.26). After staining with alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows. Specific implementation method:

[0057] Experiment 1: Experimental study on the treatment of alcohol-induced osteogenic differentiation disorder of rBMSCs by the drug of the present invention

[0058] 1. Experimental Materials

[0059] Rat bone marrow mesenchymal stem cells (rBMSCs) were purchased from Wuhan Pronocell Life Science Co., Ltd. (Cat. No.: CP-R131). The basic culture conditions of rBMSCs were Rat bone marrow mesenchymal stem cell complete medium (Cat. No.: RAXMX-80011). The conditions for osteogenic induction culture of rBMSCs are: Rat bone marrow mesenchymal stem cell osteogenic differentiation kit (Cat. No. RAXMX-90021). Anhydrous ethanol (Cat. No. C0691510123).

[0060] 2. qRT-PCR detection of osteogenic differentiation

[0061] qRT-PCR was performed to evaluate osteogenic differentiation after 6, 12, and 18 days of induction of osteogenic differentiation. Primers for Runx2, ALP, BMP2, and SP7, marker genes for osteogenic differentiation of rBMSCs stem cells, were designed by Thermo Fisher Scientific (China) Co., Ltd., and the specificity and feasibility of the primers were verified experimentally. RNA was extracted using TRIzol reagent (Invitrogen), RNA purity and concentration were measured using NanoDrop 2000 (Thermo Fisher Scientific (China) Co., Ltd.), and reverse transcription was performed using Prime-Script TM RTReagent Kit (Takara, Japan), and TB GreenTM Premix Ex Taq TM II kit (Takara, Japan), and the fluorescence quantitative instrument used was QuantStudio 3 Real-Time PCR Systems (Thermo Fisher Scientific (China) Co., Ltd.). -ΔΔCtMethods: Gene expression was normalized by the internal reference gene GAPDH. The mean of the normalized values ​​of Runx2, ALP, BMP2, and SP7 (i.e., the sum of the normalized values ​​of Runx2, ALP, BMP2, and SP7 / 4) was used to quantitatively analyze osteogenic differentiation, and the EC50 (half effective concentration) was calculated by the software as its effective concentration.

[0062] 4. Test substances

[0063] (1) Positive control group: rBMSCs + osteogenic induction culture, fresh osteogenic induction medium was replaced every 3 days, with 5 biological replicates;

[0064] (2) Blank control group: rBMSCs + basal culture medium, fresh basal culture medium was replaced every 3 days, with 5 biological replicates;

[0065] (3) Model group: rBMSCs + osteogenic induction culture + 100mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 5 biological replicates;

[0066] (4) Experimental drug group A: naringin 10 concentration gradients 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration;

[0067] (5) Experimental drug group B: 10 concentration gradients of morroniside 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration.

[0068] (6) Experimental drug group C: naringin: morroniside = 3:1, 10 concentration gradients 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration.

[0069] (7) Experimental drug group D: naringin: morroniside = 2:1, 10 concentration gradients 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration.

[0070] (8) Experimental drug group E: naringin: morroniside = 1:1, 10 concentration gradients 10-5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration.

[0071] (9) Experimental drug group F: naringin: morroniside = 1:2, 10 concentration gradients 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration

[0072] (10) Experimental drug group G: naringin: morroniside = 1:3, 10 concentration gradients 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration.

[0073] (11) Experimental drug group H: naringin: morroniside = 1:4, 10 concentration gradients 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration.

[0074] (12) Experimental drug group I: naringin: morroniside = 1:5, 10 concentration gradients 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration.

[0075] (13) Experimental drug group J: naringin: morroniside = 1:6, 10 concentration gradients 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration.

[0076] (14) Experimental drug group K: naringin: morroniside = 1:7, 10 concentration gradients 10 -5 ~10 4 μg / mL+rBMSCs+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 5 biological replicates were set for each concentration.

[0077] 5. Experimental results

[0078] Within a certain concentration range, experimental drug group A, experimental drug group B, experimental drug group C, experimental drug group D, experimental drug group E, experimental drug group F, experimental drug group G, experimental drug group H, experimental drug group I, experimental drug group J, and experimental drug group K can all improve the alcohol-induced rBMSCs osteogenic differentiation disorder. Further, by setting different concentration gradients, the EC50 concentration was calculated, as shown in Table 1 below.

[0079] Table 1 qRT-PCR detection of osteogenic differentiation and calculation of EC50 values

[0080]

[0081] 6. Experimental conclusion

[0082] Alcohol (100mmoL / L) inhibits the expression of osteogenic differentiation marker genes Runx2, ALP, BMP2 and SP7 in stem cells, leading to osteogenic differentiation disorder. High expression levels of osteogenic differentiation marker genes Runx2, ALP, BMP2 and SP7 indicate that osteogenic differentiation of cells is promoted; conversely, it indicates that osteogenic differentiation of cells is inhibited.

[0083] Within a certain concentration range, naringin and morroniside alone or in combination can improve the alcohol-induced osteogenic differentiation disorder of METTL14 non-silenced rBMSCs. The effect of naringin and morroniside combination on improving the alcohol-induced osteogenic differentiation disorder of METTL14 non-silenced rBMSCs has no significant relationship with their weight ratio.

[0084] Experiment 2: Experimental study on the drug of the present invention for treating alcohol-induced osteogenic differentiation disorder of rBMSCs with METTL14 gene silencing

[0085] 1. Experimental purpose

[0086] The purpose of this experiment is to verify the effect of the drug of the present invention on the alcohol-induced osteogenic differentiation disorder of rBMSCs with METTL14 gene silencing.

[0087] 2. Experimental Materials

[0088] Rat bone marrow mesenchymal stem cells (rBMSCs) were purchased from Wuhan Pronocell Life Science Co., Ltd. (Cat. No.: CP-R131). The basic culture conditions of rBMSCs were: Rat bone marrow mesenchymal stem cell complete medium (Cat. No.: RAXMX-80011). The conditions for osteogenic induction culture of rBMSCs are: Rat bone marrow mesenchymal stem cell osteogenic differentiation kit (Cat. No.: RAXMX-90021). Anhydrous ethanol (Cat. No.: C0691510123).

[0089] 3. Preparation of rBMSCs with METTL14 gene silencing

[0090] rBMSCs were cultured at 1×10 5 The concentration of cells / mL was inoculated in 6-well plates. siRNA or negative control oligonucleotides were synthesized by Invitrogen. siRNA (siRNA-METTL14 group) or negative control (siRNA-negative control group) oligonucleotides were transfected into BMSCs using Lipofectamine RNAiMAX reagent (MAN0007825; Invitrogen) according to the manufacturer's instructions. Subsequent experimental studies were performed 24 hours later. The sequences of the siRNAs used were:

[0091] METTL14: sense 5′-GCAGCACCUCGGUCAUUUATT-3′ and

[0092] antisense 5′-UAAAμgACCGAGGμgCμgCTT-3′.

[0093] The following grouping study was conducted, with 3 biological replicates in each group.

[0094] 4. Phenotypic verification of rBMSCs with METTL14 gene silencing

[0095] (1) Western Blot

[0096] Detection of m6A modification-related gene METTL14 protein expression. The total protein of the METTL14 gene-silenced rBMSCs prepared in the above two parts was extracted using cell lysis buffer, and the protein concentration was quantified using a protein assay kit (P0011, Shanghai Biyuntian Biotechnology Co., Ltd.). The protein samples were separated by electrophoresis on polyacrylamide gels, transferred to nitrocellulose membranes, and then blocked with 5% fat-free milk powder at 4°C overnight. The membranes were incubated with the corresponding primary antibodies (recombinant Anti-METTL14 antibody [EPR22666-22], ab300104, 1:1000); anti-GAPDH antibody (Anti-GAPDH antibody [6C5], ab8245, 1:5,000) at 4°C overnight. The membranes were washed and incubated with fluorescently labeled secondary antibodies (Goat Anti-Rabbit IgG H&L HRP, ab97051; Goat Anti-Mouse IgG H&L HRP, ab205719) at room temperature for 1.5 hours. Western blot bands were imaged by Odyssey CLx and quantified using LI-COR Image Studio software.

[0097] (2) qRT-PCR detection of METTL14 mRNA levels

[0098] The total RNA of rBMSCs with METTL14 gene silencing prepared in the above two parts was extracted. The METTL14 primer design was completed by Thermo Fisher Scientific (China) Co., Ltd., and the specificity and feasibility of the primers were verified experimentally. RNA extraction was performed using TRIzol reagent (Invitrogen), RNA purity and concentration were measured using NanoDrop 2000 (Thermo Fisher Scientific (China) Co., Ltd.), and reverse transcription was performed using Prime-Script TM RT Reagent Kit (Takara, Japan), quantitative detection using TB GreenTM Premix Ex Taq TM II kit (Takara, Japan), and the fluorescence quantitative instrument used was QuantStudio3 Real-Time PCR Systems (Thermo Fisher Scientific (China) Co., Ltd.). The 2-ΔΔCt method was used to normalize gene expression using the internal reference gene GAPDH. Figure 1 :Western Blot was used to detect the protein expression of m6A modification-related gene METTL14.

[0099] Figure 1The black strips in the middle represent the expression of METTL14 protein. The darker the color and the more obvious the strips, the higher the protein expression level. It can be seen from the figure that the expression of METTL14 protein in the siRNA-METTL14 group is significantly reduced compared with the siRNA-negative control group. GAPDH is an internal reference. See Table 2.

[0100] Table 2 Expression of METTL14 gene detected by Western Blot and qRT-PCR

[0101]

[0102] Note: Compared with the siRNA-negative control group, # P<0.05.

[0103] 5. Experimental results

[0104] Western Blot and qRT-PCR were used to detect the expression of METTL14 gene. siRNA interference with METTL14 gene expression led to a decrease in protein and mRNA expression levels in rBMSCs.

[0105] 6. Experimental conclusion

[0106] It is now known that METTL14 is a key gene regulating m6A modification, and interfering with METTL14 gene expression will lead to disordered m6A modification. The rBMSCs cell model with METTL14 gene silencing was successfully prepared, which can meet the requirements of subsequent experiments.

[0107] 7. Experimental Grouping

[0108] Referring to the results of “Experiment 1 (Experimental study on the drug of the present invention for treating alcohol-induced osteogenic differentiation disorder of rBMSCs)”, the following experiment was designed based on the drug efficacy concentration in the experimental study on the drug of the present invention for treating alcohol-induced osteogenic differentiation disorder of rBMSCs.

[0109] (1) Positive control group: rBMSCs + osteogenic induction culture, fresh osteogenic induction medium was replaced every 3 days, and 3 biological replicates were set.

[0110] (2) Blank control group: rBMSCs+basal culture medium, fresh basal culture medium was replaced every 3 days, with 3 biological replicates.

[0111] (3) Model group: rBMSCs+siRNA-METTL14+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, with 3 biological replicates.

[0112] (4) Experimental drug group A: naringin 50.128 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol. Fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0113] (5) Experimental drug group B: morroniside 36.645 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0114] (6) Experimental drug group C: naringin: morroniside = 3:1, 48.123 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0115] (7) Experimental drug group D: naringin: morroniside = 2:1, 47.003 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0116] (8) Experimental drug group E: naringin: morroniside = 1:1, 40.182 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0117] (9) Experimental drug group F: naringin: morroniside = 1:2, 36.101 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0118] (10) Experimental drug group G: naringin: morroniside = 1:3, 32.963 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0119] (11) Experimental drug group H: naringin: morroniside = 1:4, 39.107 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0120] (12) Experimental drug group I: naringin: morroniside = 1:5, 42.259 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0121] (13) Experimental drug group J: naringin: morroniside = 1:6, 47.458 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0122] (14) Experimental drug group K: naringin: morroniside = 1:7, 61.572 μg / mL + rBMSCs + siRNA-METTL14 + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0123] (15) Control-model group: rBMSCs+siRNA-negative control+osteoblast induction culture+100mmoL / L ethanol, fresh osteoblast induction culture medium was replaced every 3 days, and 3 biological replicates were set.

[0124] (16) Control-experimental drug group A: naringin 50.128 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmol / L ethanol. Fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0125] (17) Control-experimental drug group B: morroniside 36.645 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmoL / L ethanol. Fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0126] (18) Control-experimental drug group C: naringin: morroniside = 3:1, 48.123 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0127] (19) Control-experimental drug group D: naringin: morroniside = 2:1, 47.003 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0128] (20) Control-experimental drug group E: naringin: morroniside = 1:1, 40.182 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0129] (21) Control-experimental drug group F: naringin: morroniside = 1:2, 36.101 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0130] (22) Control-experimental drug group G: naringin: morroniside = 1:3, 32.963 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0131] (23) Control-experimental drug group H: naringin: morroniside = 1:4, 39.107 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0132] (24) Control-experimental drug group I: naringin: morroniside = 1:5, 42.259 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmol / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0133] (25) Control-experimental drug group J: naringin: morroniside = 1:6, 47.458 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0134] (26) Control-experimental drug group K: naringin: morroniside = 1:7, 61.572 μg / mL + rBMSCs + siRNA-negative control + osteogenic induction culture + 100 mmoL / L ethanol, fresh osteogenic induction culture medium was replaced every 3 days, with 3 biological replicates.

[0135] 8. Experimental testing

[0136] After 18 days, the following tests were performed:

[0137] (1) qRT-PCR detection of osteogenic differentiation

[0138] qRT-PCR was performed to evaluate osteogenic differentiation 18 days after induction of osteogenic differentiation. Primers for Runx2, ALP, BMP2, and SP7, marker genes for osteogenic differentiation of rBMSCs stem cells, were designed by Thermo Fisher Scientific (China) Co., Ltd., and the specificity and feasibility of the primers were verified experimentally. RNA was extracted using TRIzol reagent (Invitrogen), RNA purity and concentration were measured using NanoDrop 2000 (Thermo Fisher Scientific (China) Co., Ltd.), and reverse transcription was performed using Prime-Script TM RTReagent Kit (Takara, Japan), and TB GreenTM Premix Ex Taq TM II kit (Takara, Japan), and the fluorescence quantitative instrument used was QuantStudio 3 Real-Time PCR Systems (Thermo Fisher Scientific (China) Co., Ltd.). -ΔΔCt Methods: Gene expression was normalized by the internal reference gene GAPDH.

[0139] (2) Alizarin red staining

[0140] After inducing osteogenic differentiation for 18 days, alizarin red staining was performed to evaluate the formation of calcified nodules. The cells were fixed with 4% paraformaldehyde for 15 minutes, and stained with alizarin red staining solution (Beijing Solebow Technology Co., Ltd., Cat. No.: G1450) at room temperature for 5 minutes, then the staining solution was removed and the cells were rinsed with deionized water 3-4 times. The alizarin red-positive area was analyzed using ImageJ software (NIH), and the ratio of the alizarin red-positive area to the total area was expressed.

[0141] 9. Experimental results

[0142] The experimental results are shown in Table 3. Figure 2 — Fig. 27 .

[0143] Compared with the positive control group, alcohol inhibited the expression of osteogenic differentiation marker genes Runx2, ALP, BMP2 and SP7 in the model group, leading to osteogenic differentiation disorder. High expression levels of osteogenic differentiation marker genes Runx2, ALP, BMP2 and SP7 indicate that osteogenic differentiation of cells is promoted; conversely, it indicates that osteogenic differentiation of cells is inhibited.

[0144] It is now known that the METTL14 gene is a key gene regulating m6A modification, and interfering with the expression of the METTL14 gene will lead to disordered m6A modification.

[0145] In control-experimental drug groups A, B, C, D, E, F, G, H, I, J, and K, the METTL14 gene was not silenced and the m6A modification was normal;

[0146] The METTL14 gene was silenced and the m6A modification was disordered in the experimental drug groups A, B, C, D, E, F, G, H, I, J, and K.

[0147] Compared with the model group, experimental drug group F, experimental drug group G, and experimental drug group H could improve the alcohol-induced osteogenic differentiation disorder of rBMSCs with METTL14 gene silencing to some extent, while other drug groups (experimental drug groups A, B, C, D, E, I, J, and K) did not significantly improve the alcohol-induced osteogenic differentiation disorder of rBMSCs with METTL14 gene silencing.

[0148] Compared with the control-model group, the control-experimental drug groups (control-experimental drug A, B, C, D, E, F, G, H, I, J, and K groups) could significantly improve the alcohol-induced osteogenic differentiation disorder of rBMSCs in which the METTL14 gene was not silenced.

[0149] In summary, experimental drug groups F, G, and H can target m6A modification to treat alcohol-induced stem cell osteogenic differentiation disorder with METTL14 gene silencing, while experimental drug groups A, B, C, D, E, I, J, and K do not have this function.

[0150] Table 3 Expression of osteogenic differentiation marker genes Runx2, ALP, BMP2, and SP7 detected by qRT-PCR

[0151]

[0152]

[0153] Note: Compared with the model group, * P<0.05; compared with the control-model group, # P<0.05; compared with the positive control group, & P<0.05.

[0154] Figure 2 Shown: In the positive control group (0.92±0.07), the calcium salt deposition in the mineralized nodules of cells appeared orange-red after staining with Alizarin red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0155] Figure 3 Shows: In the blank control group (0.09±0.05), after staining with Alizarin red, no cell mineralized nodules were observed, where calcium salt deposition appeared orange-red, and no significant mineralized nodules were formed.

[0156] Figure 4Show: Model group (0.39±0.19), after Alizarin red staining, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0157] Figure 5 Display: Experimental drug group A: naringin (0.41±0.21), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0158] Figure 6 Display: Experimental drug group B: Morroniside (0.40±0.22), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0159] Figure 7 Display: Experimental drug group C: naringin: morroniside = 3:1 (0.38±0.19), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0160] Figure 8 Display: Experimental drug group D: naringin: morroniside = 2:1 (0.39±0.21). After staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0161] Fig. 9 Display: Experimental drug group E: naringin: morroniside = 1:1 (0.37±0.22). After staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0162] Fig.10 Display: Experimental drug group F: naringin: morroniside = 1:2 (0.79±0.22), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0163] Fig.11 Display: Experimental drug group G: naringin: morroniside = 1:3 (0.89±0.19), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0164] Fig.12Display: Experimental drug group H: naringin: morroniside = 1:4 (0.71±0.23). After staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0165] Fig.13 Display: Experimental drug group I: naringin: morroniside = 1:5 (0.41±0.20), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0166] Fig.14 Display: Experimental drug group J: naringin: morroniside = 1:6 (0.39±0.19), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0167] Fig.15 Display: Experimental drug group K: naringin: morroniside = 1:7 (0.41±0.24). After staining with Alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0168] Fig.16 Display: Control-model group: (0.56±0.24), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0169] Fig.17 Display: Control-experimental drug group A: naringin (0.79±0.19), after staining with alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0170] Fig.18 Display: Control-experimental drug group B: Morroniside (0.83±0.18), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0171] Fig.19 Display: Control-experimental drug group C: naringin: morroniside = 3:1 (0.69±0.17). After staining with Alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0172] Fig. 20Display: Control-experimental drug group D: naringin: morroniside = 2:1 (0.89±0.20), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0173] Fig.21 Display: Control-experimental drug group E: naringin: morroniside = 1:1 (0.86±0.21). After staining with Alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0174] Fig. 22 Display: Control-experimental drug F group: naringin: morroniside = 1:2 (0.87±0.19), after staining with Alizarin red, the calcium salt deposition in the cell mineralized nodules appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0175] Fig.23 Display: Control-experimental drug group G: naringin: morroniside = 1:3 (0.86±0.21). After staining with Alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0176] Fig.24 Display: Control-experimental drug group H: naringin: morroniside = 1:4 (0.90±0.26). After staining with Alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0177] Fig.25 Display: Control-experimental drug group I: naringin: morroniside = 1:5 (0.89±0.25), after staining with Alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious), and the mineralized nodules are shown by the black arrows.

[0178] Fig.26 Display: Control-experimental drug group J: naringin: morroniside = 1:6 (0.889±0.19). After staining with Alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0179] Fig. 27 Display: Control-experimental drug K group: naringin: morroniside = 1:7 (0.89±0.26). After staining with Alizarin red, the calcium salt deposition in the mineralized nodules of the cells appeared orange-red (the original image is a color image, and the coloring is more obvious). The mineralized nodules are shown by the black arrows.

[0180] Note: The numbers in the brackets above indicate the proportion of the orange-red area of ​​calcium salt deposits in the mineralized nodules of cells stained with alizarin red to the total area.

[0181] The results of Alizarin red staining indicated that experimental drug group F, experimental drug group G, and experimental drug group H could improve the osteogenic differentiation disorder of rBMSCs induced by alcohol with METTL14 gene silencing to some extent.

[0182] 10. Experimental Conclusion

[0183] It is now known that METTL14 is a key gene regulating m6A modification. Interference in METTL14 gene expression will lead to disordered m6A modification. In the control-experimental drug A, B, C, D, E, F, G, H, I, J, K groups, the METTL14 gene was not silenced, and m6A modification was normal; in the experimental drug A, B, C, D, E, F, G, H, I, J, K groups, the METTL14 gene was silenced, and m6A modification was disordered. The high expression levels of osteogenic differentiation marker genes Runx2, ALP, BMP2, and SP7 indicate that osteogenic differentiation of cells is promoted; conversely, osteogenic differentiation of cells is inhibited.

[0184] When the METTL14 gene is not silenced and the m6A modification is normal, within a certain concentration range, naringin and morroniside used alone or in combination can improve the alcohol-induced osteogenic differentiation disorder of rBMSCs. There is no significant relationship between the improvement of alcohol-induced osteogenic differentiation disorder of rBMSCs by the combination of naringin and morroniside and their weight ratio.

[0185] When the METTL14 gene is silenced and the m6A modification is abnormal, only the naringin and morroniside composition composed of a specific weight ratio (naringin: morroniside = 1:2, 1:3, 1:4) can target m6A modification to improve the alcohol-induced rBMSCs osteogenic differentiation disorder with METTL14 gene silencing, while the naringin and morroniside compositions composed of other weight ratios did not improve the osteogenic differentiation disorder. In other words, the improvement of the naringin and morroniside composition in improving the alcohol-induced rBMSCs osteogenic differentiation disorder with METTL14 gene silencing is closely related to its weight ratio.

Claims

1. A drug, characterized in that The medicine is prepared from the following raw materials in parts by weight: naringin and morroniside.

2. The drug according to claim 1, characterized in that The drug is a drug for treating alcohol-induced osteogenic differentiation disorder of bone marrow mesenchymal stem cells in which the METTL14 gene is not silenced, and is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 0.33-7 parts by weight of morroniside.

3. The drug according to claim 2, characterized in that The medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 2 parts by weight of morroniside.

4. The drug according to claim 2, characterized in that The medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 3 parts by weight of morroniside.

5. The drug according to claim 1, characterized in that The drug is a drug for treating alcohol-induced osteogenic differentiation disorder of bone marrow mesenchymal stem cells with METTL14 gene silencing. The drug is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 2 to 4 parts by weight of morroniside.

6. The drug according to claim 5, characterized in that The medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 2 parts by weight of morroniside.

7. The drug according to claim 5, characterized in that The medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 3 parts by weight of morroniside.

8. The drug according to claim 5, characterized in that The medicine is prepared from the following raw materials in parts by weight: 1 part by weight of naringin and 4 parts by weight of morroniside.

9. The drug according to any one of claims 1, 2 or 5, characterized in that The medicine is prepared into tablets, pills, powders, hard capsules, soft capsules, granules and oral liquids by conventional methods in pharmaceutical preparations.

10. Use of 1 part by weight of naringin and 2-4 parts by weight of morroniside as raw materials in the preparation of a drug for treating alcohol-induced osteogenic differentiation disorder of bone marrow mesenchymal stem cells with METTL14 gene silencing.

Images