In-vitro proliferation method of bone marrow mesenchymal stem cells

Abstract

The invention relates to the technical field of bone marrow mesenchymal stem cell proliferation, and particularly discloses an in-vitro proliferation method of bone marrow mesenchymal stem cells, which comprises the following steps: S1, preparing rat bone marrow mesenchymal stem cells, performing constant-temperature culture on the rat bone marrow mesenchymal stem cells under the condition of 5% CO2 at 36-37 DEG C, and then performing passage; s2, taking third-generation rat bone marrow mesenchymal stem cells, adjusting the cell concentration to 1 * 10 < 7 > cells / L, culturing in the basic culture medium, and adding a mulberry leaf extract into the basic culture medium; and S3, adding a CCK-8 reagent, incubating in a CO2 incubator in a dark place for 3 hours, and detecting the proliferation condition of the rat bone marrow mesenchymal stem cells. When the mulberry leaf extract is applied to a basic culture medium, positive regulation and control of cell proliferation and negative regulation and control of a cell apoptosis process can be realized, so that the mulberry leaf extract is applied to preparation of a medicine for preventing and treating periodontitis; and a new experimental basis and a new theoretical basis are provided for research, development and clinical application of the mulberry leaf extract-based periodontal prevention and treatment medicine.

Description

Technical Field

[0001] The present invention relates to the technical field of bone marrow mesenchymal stem cell proliferation, and in particular to an in vitro proliferation method of bone marrow mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSC) are an important member of the stem cell family, originating from the early mesoderm and ectoderm. MSCs can differentiate into osteoblasts, chondroblasts and other cells, and are therefore widely used in cell-mediated anti-tumor gene therapy, hematopoietic stem cell transplantation, tissue damage repair and autoimmune diseases.

[0003] Periodontitis is a chronic infectious disease of periodontal tissues caused by subgingival plaque stimulation, and is one of the main factors leading to periodontal tissue destruction and loss of function. Periodontal pathogens attach to the surface of teeth, and then form dental plaque, which causes changes in microcirculation of tissues around teeth, activation of host immune cells and release of inflammatory factors, leading to gingival inflammation, periodontal pocket formation and alveolar bone resorption, and ultimately leading to loose and falling teeth. According to the "Fourth National Oral Health Epidemiological Survey Report" published in 2017, the detection rate of dental tartar in the 35-44-year-old population of Chinese residents is 96.7%, and the rate of gingival bleeding is 87.4%, which is the main factor threatening the oral health of the Chinese people and causing tooth loss. Worldwide, the prevalence of periodontitis is 45%-50%, among which severe periodontitis affects the oral health of 11.2% of the world's population and is also one of the sixth most common human diseases in the world. Periodontal pathogens play a key role in the pathogenesis of periodontal disease, but the progression of periodontal tissue destruction depends on the host's immune response. The key proinflammatory factors and chemokines in the immune system are IL-1β, IL-6, and MMP9. Since they promote leukocyte extravasation and induce the release of matrix metalloproteinases, especially the release and activation of MMP9, these mediators are considered to be closely related to the progression of periodontal disease. MMP9 can also destroy the collagen layer on the bone surface, promote the formation of osteoclasts, and play an important role in the progression of periodontitis. At the same time, in inflammatory tissues, IL-6 can stimulate the secretion of RANKL and reduce the expression of OPG, leading to bone destruction. Dental plaque is the driving factor of periodontitis. The main pathogen of chronic periodontitis is Porphyromonas gingivalis. At the same time, the local microenvironment of periodontitis is an inflammatory plus hypoxic environment. Inflammation plays a key role in the physiological response to hypoxic stress. Cellular hypoxia can trigger the expression of several inflammatory mediators, send tissue damage signals, and initiate survival response mediators.

[0004] At present, there are various treatments for periodontal disease, mainly based on conventional treatments that rely on periodontal basic treatments supplemented by systemic or local application of antibiotics and other drugs. The basic treatment is mainly based on the etiological treatment of mechanically removing plaque and subgingival calculus from the periodontal pockets such as scaling and root planing. Only in severe cases, periodontal regenerative surgery and other methods are used to reconstruct the defective periodontal tissue. Conventional therapy has a certain effect on the control of periodontitis, but it cannot regenerate and repair the defective periodontal tissue. At the same time, systemic application of antibiotics can also lead to side effects such as dysbiosis and the production of drug-resistant strains. In recent years, due to the advantages of Chinese herbal medicines such as small side effects, wide biological activity and low price, many scholars at home and abroad have conducted extensive exploration in the field of traditional Chinese medicine for the prevention and treatment of periodontitis. As chemical substances derived from plants, plant active ingredients have great potential for use as drugs, and a large number of studies have reported various important pharmacological activities of plant active ingredients. At the same time, more and more scholars have advocated the use of effective ingredients from natural plants to prevent and treat periodontitis, which has become a potential new periodontal prevention and treatment strategy.

[0005] To this end, the present invention aims to provide an in vitro proliferation method of bone marrow mesenchymal stem cells, which can be applied to regenerate and repair defective periodontal tissues and solve the problems in the above-mentioned prior art. Summary of the invention

[0006] The purpose of the present invention is to solve the technical problems raised in the above background technology and to provide an in vitro proliferation method of bone marrow mesenchymal stem cells.

[0007] The above-mentioned purpose of the present invention is achieved like this:

[0008] The present invention provides, on one hand, a method for in vitro proliferation of bone marrow mesenchymal stem cells, comprising the following steps:

[0009] S1. preparing rat bone marrow mesenchymal stem cells, culturing them at a constant temperature of 36°C to 37°C under a 5% CO2 condition, and then passage them;

[0010] S2. Take the third generation of rat bone marrow mesenchymal stem cells and adjust the cell concentration to 1×10 7 After the cells were grown in the basal medium, mulberry leaf extract was added to the basal medium.

[0011] S3, then add CCK-8 reagent and incubate in a CO2 incubator in the dark for 3 hours to detect the proliferation of rat bone marrow mesenchymal stem cells.

[0012] Furthermore, the amount of mulberry leaf extract added in step S2 is 10 mg / L-150 mg / L.

[0013] Furthermore, the amount of the mulberry leaf extract is 10 mg / L.

[0014] Furthermore, the concentration of the mulberry leaf extract is 1-100 μg / mL.

[0015] Another aspect of the present invention provides an application of mulberry leaf extract in the proliferation of bone marrow mesenchymal stem cells.

[0016] Another aspect of the present invention provides an in vitro proliferation method of bone marrow mesenchymal stem cells for use in preparing a drug for preventing and treating periodontitis.

[0017] The difficulty and significance of the technical problem solved by the present invention are:

[0018] Although the periodontal disease prevention and treatment drugs in the current prior art can play a certain therapeutic effect, they are still very limited in the process of periodontitis prevention and treatment. Although they can play a role in plaque control, they cannot restore the damaged soft and hard tissues. At the same time, the commonly used periodontal tissue regeneration treatment techniques in clinical practice include bone transplantation and guided tissue regeneration, but they have failed to achieve true periodontal tissue regeneration, and the repair and regeneration of patients' periodontal tissues are still facing major challenges. Therefore, the application of simple antibacterial drugs or anti-inflammatory drugs in the prevention and treatment of periodontitis has certain limitations. If there are drugs that can simultaneously regulate the inflammatory pathway, the hypoxia pathway and promote tissue regeneration, this drug is the ideal drug for the prevention and treatment of periodontitis. Natural drugs have gradually become a research hotspot in recent years due to their small adverse reactions, low cost and wide acceptable range. Screening effective ingredients for antibacterial, anti-inflammatory and alveolar bone regeneration from natural drugs has become one of the new ways to develop drugs for the prevention and treatment of periodontitis. The invention of this application has been verified through research and experiments, and provides the application of mulberry leaf extract in the in vitro proliferation of bone marrow mesenchymal stem cells and the prevention and treatment of periodontitis. It can establish a microenvironment that is conducive to the repair and regeneration of periodontal tissue to prevent and treat periodontitis, and provide an experimental basis and theoretical basis for the research, development and clinical application of periodontal prevention and treatment drugs based on mulberry leaf extract.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] In the in vitro proliferation method of bone marrow mesenchymal stem cells provided by the present invention, mulberry leaf extract is applied to the basal culture medium, which can achieve positive regulation of cell proliferation and negative regulation of cell apoptosis process, so that the mulberry leaf extract is applied to the preparation of drugs for preventing and treating periodontitis, providing a new experimental basis and a new theoretical basis for the research, development and clinical application of periodontal prevention and treatment drugs based on the development of mulberry leaf extracts. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The daily body weight and increase of rats during the experimental period in the embodiment of the present invention ((A) daily body weight change of rats during the experimental period; (B) daily body weight increase of rats during the experimental period);

[0022] Figure 2 This is a rat periodontitis modeling in an embodiment of the present invention ((A) modeling schematic diagram; (B) X-ray inspection of whether the modeling is successful, the left picture is a normal rat image, and the right picture is a model group rat image);

[0023] Figure 3 Gingival bleeding index, probing depth and tooth mobility of rats with periodontitis in the embodiment of the present invention ((A) daily gingival bleeding index (GBI: 0-5) of rats during the experimental period; (B) daily probing depth (PPD) of rats during the experimental period; (C) PPD difference analysis of rats in each group at the end of the experiment; (D) M1 looseness (TM: 0-3) difference analysis of rats in each group at the end of the experiment. Data are expressed as mean ± standard deviation (n = 6). Different superscript letters represent significant differences among groups (P < 0.05));

[0024] Figure 4 3D and cross-sectional Micro-CT images of the right maxillary bone in the embodiment of the present invention (CEJ-ABC distances of the mesial, distal and central sites are calculated in the 3D image. The sagittal, coronal and horizontal planes are in sequence);

[0025] Figure 5 The alveolar bone parameters in the embodiment of the present invention ((A) CEJ-ABC; (B) BMD; (C) BV / TV (%); (D) Tb.Th; (E) Tb.N; (F) Tb.Sp. The data are expressed as mean ± standard deviation (n = 6). Different superscript letters represent significant differences among the groups (P < 0.05));

[0026] Figure 6 The immunohistochemical staining results and quantification of Ocn, BMP2 and Runx2 in the embodiment of the present invention ((A) Immunohistochemical staining results of Ocn, (B) BMP2 and (C) Runx2. (D) Quantitative analysis of Ocn (E) BMP2 and (F) Runx2. Data are expressed as mean ± standard deviation (n = 6). Different superscript letters represent significant differences among the groups (P < 0.05));

[0027] Figure 7 The staining and inflammation scores in the present invention ((A) HE staining: BG group, the gingival epithelium is intact, the connective tissue is dense, and there is almost no inflammatory cell infiltration; the epithelial integrity of the PG group is destroyed, a large number of inflammatory cells infiltrate, the alveolar bone height is significantly reduced, and the alveolar ridge top is irregularly destroyed. (B) HE staining inflammation score. The data are expressed as mean ± standard deviation (n = 6). Different superscript letters represent significant differences between the groups (P < 0.05));

[0028] Figure 8The periodontal tissue staining in the embodiment of the present invention. (A) Sirius red staining; (B) Masson staining; (C) Trap staining; (D) Measurement of collagen content per Sirius red staining unit; (D) Measurement of collagen content per Masson staining unit; (E) Trap positive cell count. Data are expressed as mean ± standard deviation (n = 6). Different superscript letters represent significant differences between groups (P < 0.05). DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0030] The following is a detailed description of the implementation of the present invention in conjunction with specific embodiments. For those skilled in the art, the specific meanings of the following terms can be understood according to specific circumstances, and should not be understood as limitations on this patent.

[0031] With reference to the drawings, the following are preferred embodiments of the present invention.

[0032] Embodiment: The embodiment of the present invention provides a method for in vitro proliferation of bone marrow mesenchymal stem cells, the method comprising the following steps:

[0033] S1. preparing rat bone marrow mesenchymal stem cells, culturing them at a constant temperature of 36°C to 37°C under a 5% CO2 condition, and then passage them;

[0034] S2. Take the third generation of rat bone marrow mesenchymal stem cells and adjust the cell concentration to 1×10 7 After the cells were grown in a basal medium, 10 mg / L of mulberry leaf extract with a concentration of 1-100 μg / mL was added to the basal medium;

[0035] S3, then add CCK-8 reagent and incubate in a CO2 incubator in the dark for 3 hours to detect the proliferation of rat bone marrow mesenchymal stem cells.

[0036] In the scheme of this embodiment, the application of mulberry leaf extract in the proliferation of bone marrow mesenchymal stem cells is mainly provided. In addition, the in vitro proliferation method of bone marrow mesenchymal stem cells of the present invention is used in the preparation of drugs for preventing and treating periodontitis.

[0037] The following is a specific experiment of the present invention:

[0038] 1 Experimental content and methods

[0039] 1.1 Experimental subjects

[0040] 1.1.1 Experimental animals

[0041] Specific pathogen free (SPF) rats used in this experiment were purchased from the Animal Experiment Center of Xinjiang Medical University. Sprague-Dawley (SD) male rats aged 4-6 weeks were used, with a body weight ranging from 100g to 150g. The experimental animal production license number is SCXK (Xin) 2020-0002.

[0042] 1.1.2 Experimental reagents and consumables

[0043] Mulberry leaf extract was provided by the School of Pharmacy of Xinjiang Medical University; minocycline hydrochloride (CAS No.: 13614-98-7) was purchased from Shanghai Yuanye Biological Co., Ltd.; glycerol (CAS No.: 56-81-5) was purchased from Tianjin Damao Chemical Reagent Factory; EP tubes, centrifuge tubes, and disposable pipette tips of different specifications were purchased from Wuxi Nice Life Science Co., Ltd. (China); sealing film (catalog number: PM-996) was purchased from Parafilm (USA); disposable filters (0.22μm) were purchased from Millipore Corporation (USA); 1mL and 5mL disposable syringes were purchased from Kangdeli Enterprise Development Group Co., Ltd. (China).

[0044] 1.1.3 Experimental periodontitis modeling consumables

[0045] SD rats, bedding and feed were purchased from the Animal Experiment Center of Xinjiang Medical University; IVC (Individual ventilated cages) feeding system, IVC mouse cage system (rat) and rat dissection board were provided by the Animal Experiment Center of Xinjiang Military Command General Hospital; 4-0 ligature silk thread was purchased from Johnson & Johnson (China) Medical Device Co., Ltd.; titanium alloy microsurgical instrument kit was purchased from Dongyu Medical; atropine hydrochloride injection was purchased from Shanghai Quanyu Biotechnology (Zhumadian) Animal Pharmaceutical Co., Ltd.; Lumianning (xylazine hydrochloride injection) and Luxingning (benzoxazole hydrochloride injection) were purchased from Jilin Huamu Animal Health Products Co., Ltd.; Shutai 50 (tiletamine hydrochloride and zolazepam hydrochloride for injection) was purchased from Virbac Co., Ltd. (France).

[0046] 1.1.4 Quantitative Real-time PCR (RT-qPCR) reagents

[0047] RNAstore sample preservation solution (DP408-02) was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; Trizol RNA isolation reagent was purchased from Invitrogen (USA); anhydrous ethanol, 75% ethanol, chloroform, and isopropanol were purchased from Tianjin Damao Chemical Reagent Factory (China); RNase, DNase and DNA Away, and DEPC water were purchased from Bio-Tech Biotechnology Co., Ltd. (China); TB Premix Ex Taq TM II(Tli RNaseH Plus), PrimeScript TM RTreagentKitwith gDNA Eraser (PerfectReal Time) was purchased from Takara Biomedical Technology Co., Ltd. (Japan)

[0048] 1.1.5 Enzyme linked immunosorbent assay (ELISA) reagents

[0049] Disposable vacuum negative pressure serum collection tubes and disposable moisture-absorbing paper tips were purchased from Hebei Chaoran Medical Instrument Co., Ltd. (China); sealing membranes and absorbent paper were purchased from Beijing Solebaugh Technology Co., Ltd. (China); RatIL6 ELISA KIT (Cat. No.: CSB-E04640r), Rat MMP-9 ELISA Kit (Cat. No.: CSB-E08008r), and Rat PTGS2 ELISA Kit (Cat. No.: CSBE13399r) were all purchased from Wuhan Huamei Bioengineering Co., Ltd. (China).

[0050] 1.1.6 Reagents and consumables related to histopathological analysis

[0051] Anhydrous ethanol, 95% ethanol, 75% ethanol, and xylene were purchased from Tianjin Damao Chemical Reagent Factory (China); EDTA decalcification solution (pH 7.2) (Cat. No. E1171), neutral formalin fixative (10%, RNase free) (Cat. No. G2162), modified picrosirius red staining kit (Cat. No. G1472), neutral gum (Cat. No. G8590), EDTA antigen retrieval solution (50X, C1034), PBS phosphate buffer powder (Cat. No. P1010), and 5% BSA blocking solution (Cat. No. SW3015) were purchased from Beijing Solebow Technology Co., Ltd. (China); DAB color development kit (Cat. No. ZLI-9017), eosin staining solution (Cat. No. Z LI-9613) and hematoxylin staining solution (catalog number: ZLI-9610) were purchased from Beijing Zhongshan Jinqiao Biotechnology Co., Ltd. (China); tartrate-resistant acid phosphatase staining kit (catalog number: 387A) and MASSON trichrome staining kit (catalog number: 1.00485) were purchased from Sigma (USA); paraffin slices and 819 blades were purchased from Leica (Germany); anti-slip slides and cover slips were purchased from Jiangsu Shitai Experimental Instrument Co., Ltd. (China); Ptgs2 Antibody (catalog number: AF7003), TNF alpha Antibody (catalog number: AF7014), IL1 beta Antibody (catalog number: AF5103), IL6 Antibody (catalog number: DF6087), BMP2 Antibody (catalog number: AF5163), Ocn Antibody (catalog number: DF12303), Opg Antibody (catalog number: DF6824), Goat AntiRabbit IgG HRP (catalog number: S0001) were purchased from Affinity Biosciences (USA); BAX antibody (catalog number: 50599-2-Ig) and BCL2 antibody (catalog number: 26593-1-AP) were purchased from Proteintech Group, Inc (USA).

[0052] 1.1.7 Large experimental equipment

[0053] Constant temperature water bath (Sigma, USA), vertical flow clean bench (Shanghai Boxun Industrial Co., Ltd. Medical Equipment Factory, model: SW-CJ-2FD), vortexer (Qilin Bell Instrument Manufacturing Co., Ltd., VORTEX-6), low-speed desktop centrifuge (Sigma, USA), low-temperature high-speed centrifuge (Sigma, USA), full-wavelength microplate reader (Thermo, USA), 4°C refrigerator (Haier, China), -20°C low-temperature refrigerator (Haier, China), -80°C ultra-low temperature refrigerator (Thermo, USA), electronic scale (YB202N, Shanghai Yitai Electronic Technology Co., Ltd.), HistoCore Arcadia automatic tissue embedding machine (Leica, Germany), ultrathin slicer (Leica, Germany), biological microscope (Leica, Germany, M320 F12), dissecting microscope (ZESS, Germany), polarized light microscope NIKON Eclipse ci (Nikon, Japan), RNA clean bench (Rabet, China), digital display constant voltage and constant current electrophoresis instrument (BIO-RAD, USA), gel imaging system (BIORAD, USA), PCR instrument (BIO-RAD, USA), fluorescence quantitative PCR amplification instrument (BIO-RAD, USA), fluorescence quantitative PCR amplification instrument (ABI, USA), X-ray machine (Bruker MS FX Pro, USA), SCANCO MEDICALμCT100 (Switzerland). All the above instruments were provided by the Clinical Research Institute of the First Affiliated Hospital of Xinjiang Medical University and the Central Laboratory of Xinjiang Medical University.

[0054] 1.2 Experimental methods

[0055] 1.2.1 Animals and experimental design

[0056] The SD rats involved in this experiment were supervised by the Animal Experiment Ethics Committee of the Xinjiang Military General Hospital. All methods were performed in accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals, compiled by the National Academy of Sciences and published by the National Institutes of Health. Considering the risk of rat loss and modeling success rate during the experiment, this experiment referred to a number of studies at home and abroad to determine the sample size to provide 80% power to identify significant differences between groups with a 95% confidence interval (α = 0.05), and the sample size of each group was determined to be 6. Referring to the modeling success and drug intervention time, the experimental period was determined to be 14 days. A total of 7 groups were set up in the experiment, that is, a total of 42 male SD rats were used, and the groups were as follows: ① Blank control group (BG): no drug application, no ligation; ② Periodontitis group (PG): no drug application, simple ligation; ③ Glycerol solvent group (GG): topical application of glycerol (solvent for dissolving mulberry leaf extract, 50% glycerol), ligation; ④ Positive drug control group (Control positive group, CG): topical application of Palio (2% minocycline hydrochloride), ligation; ⑤ High concentration foliummori group (HM): topical application of 100μg / mL mulberry leaf extract, ligation; ⑥ Medium concentration foliummori group (MM): topical application of 10μg / mL mulberry leaf extract, ligation; ⑦ Low concentration folium mori group (LM): topical application of 1μg / mL mulberry leaf extract, ligation. Periodontitis modeling method: rats in each group were deprived of food and water for 6 hours before surgery. For rats in each group except the blank group, Lumianning and Shutai were slowly injected into the hind leg muscles of the rats at a ratio of (V:V=1:1) at a dose of 10 mg / kg to achieve general anesthesia. After complete anesthesia, silk thread ligation was used to induce periodontitis. 4-0 sterile silk thread was used to wrap around the cervical part of the first molar of the right maxillary of the rat, and the silk thread was allowed to enter the gingival sulcus of the rat as much as possible. At the same time, the drug, solvent and positive control groups began to apply the corresponding drugs locally, such as Figure 1As shown. Before daily intervention, check whether the silk thread is detached or loose, and re-ligate if necessary. After the drug intervention is completed, the rats are deprived of food and water for half an hour. During the experiment, the rats were kept in the Animal Experiment Center of the General Hospital of Xinjiang Military Region. The breeding temperature was 25±3℃, the humidity was controlled in the range of 45-70%, and they were free to eat and drink pure water. The bedding was changed every four days, and the laboratory and cages were disinfected every week. Before the experiment, the rats needed to be adaptively fed for 4-6 days, and the next experiment was carried out after confirming that there were no abnormalities. After the experiment started, the animals were weighed daily, and periodontal-related data were measured. At the same time, the rats' food and water intake and mental state were closely monitored to keep the laboratory environment clean.

[0057] 1.2.2 Preparation of drugs

[0058] Source of mulberry leaf extract: provided by the School of Pharmacy of Xinjiang Medical University. Preparation of drug intervention group: GG (solvent group): 1 mL of 50% glycerol solution was drawn and placed in an EP tube and stored at 4°C; HM (high concentration mulberry leaf extract group): 10 mg of mulberry leaf extract powder was dissolved in 10 mL of 50% glycerol to make a mother solution, mixed, and diluted to a concentration of 100 μg / mL with 50% glycerol, and stored at 4°C for use; MM (medium concentration mulberry leaf extract group): 100 μL of high concentration extract solution was drawn, diluted to 1 mL with 50% glycerol, mixed, and the concentration was 10 μg / mL, and stored at 4°C for use; LM (low concentration mulberry leaf extract group): 100 μL of medium concentration extract solution was drawn, diluted to 1 mL with 50% glycerol, mixed, and the concentration was 1 μg / mL, and stored at 4°C for use; all the above drugs were prepared within 1 hour before intervention.

[0059] 1.2.3 Drug intervention

[0060] After the rats were ligated, the same experimenter performed intervention according to the grouping requirements. After measuring the data daily, the drug solution was injected into the gingival sulcus of the rats using a periodontal drug delivery device to make it evenly adhere to the gingival sulcus. The rats were released after 2 minutes of maintenance. After that, they were fasted and deprived of water for 1 hour to increase the drug residence time. The intervention was performed once a day for 14 days. The modeling method was as follows: Figure 2 As shown in (A) in .

[0061] 1.2.4 Measurement of basic periodontal indicators

[0062] 1.2.4.1 Gingival bleeding index (GBI)

[0063] Before daily drug intervention, the same professionally trained experimenter used an adapted probe with a tip radius of 0.2 mm to probe the gingival sulcus or periodontal pocket area of ​​the ligated molars and observed and scored whether there was bleeding. The scoring criteria were as follows: 0 points: gingival margin and gingival papilla (GM&P) were healthy, and no bleeding was observed after slight probing; 1 point: GM&P was mildly inflamed, and no bleeding was observed after slight probing; 2 points: GM&P was mildly inflamed, and color changes, no edema, and punctate bleeding could be observed after slight probing; 3 points: GM&P was moderately inflamed, with color changes, mild edema, and bleeding after slight probing, but blood did not overflow from the gingival sulcus; 4 points: GM&P was severely inflamed, with color changes, severe edema, and bleeding after slight probing, and blood overflowed from the gingival sulcus; 5 points: GM&P was severely inflamed, with color changes, severe edema, bleeding after slight probing or spontaneous bleeding, ulcers, and blood could be observed flowing out of the gingival sulcus.

[0064] 1.2.4.2 Probing pocket depth (PPD)

[0065] In this experiment, since the clinical periodontal probe cannot be inserted into the periodontal pocket of rats, we need to improve the periodontal probe. The tip radius is reduced to 0.2 mm, and the original scale is restored. After daily GBI measurement, the experimenter then measured the gingival sulcus / periodontal pocket depth at three sites (mesiocheir, distocheir, and mid-buccal) of the right maxillary first molars in each group and recorded the data.

[0066] 1.2.5 Imaging examination

[0067] On the 14th day after modeling, the rats in the blank control group and the model group were anesthetized and the right maxillary bones of the rats were photographed using an X-ray machine. The X-ray photos were analyzed. If the model group showed obvious alveolar bone absorption in the modeling area compared with the blank control group, it proved that the periodontitis modeling was successful. Figure 2 As shown in (B) in .

[0068] 1.2.6 Rat killing and gross specimen collection

[0069] (1) After the 14-day experiment was completed and the model was successfully established, the rats were killed and blood was collected from the abdominal aorta after general anesthesia. The collected blood was stored in a serum collection tube. As much blood as possible was collected to avoid blood retention in the alveolar bone and other tissues. After the blood in the blood collection tube coagulated, it was centrifuged at 1500 rpm at 4°C for 15 min to obtain serum. The serum was aliquoted into sterile EP tubes, sealed, and placed in a -80°C refrigerator for later use.

[0070] (2) After the rats were euthanized, the buccal surface of the right maxillary first molar of the rats was wiped with a sterile cotton ball and kept moist. Two No. 40 standardized sterilized paper tips were inserted into the periodontal pockets at the mesial and distal positions until slight resistance was encountered. After standing for 15 seconds, they were taken out and placed in an EP tube containing 1 ml of sterile PBS (if there was blood on the paper tip, it was discarded and taken out again), and then frozen at -20°C for later use;

[0071] (3) After the rats were euthanized, sterile instruments were used to immediately dissect the right maxillary bone of the rats, which contained tissues such as the gums, dentition, and alveolar bone. Two rats were randomly selected from each group, and all tissue specimens were carefully peeled off using sterile microscopic instruments. The gingival tissue and jaw bone tissue at the buccal gingival margin were rinsed with 4°C saline, and stored in pre-labeled cryopreservation tubes containing RNAstore sample preservation solution and frozen at -80°C.

[0072] (4) After the jaw tissue samples were trimmed appropriately, they were rinsed with 4°C saline, fixed in 4% paraformaldehyde for 24 h, rinsed overnight under running water, and then stored in 75% ethanol for Micro-CT detection;

[0073] (5) After the Micro CT test, the sample was placed in a 5% ethylenediaminetetraacetic acid (EDTA) decalcification solution for decalcification. The decalcification solution should completely cover the sample, and the volume ratio of the decalcification solution to the sample should be at least 10:1. The decalcification solution was changed every three days until there was no obvious resistance when the decalcified tissue was pierced with a pin, which was considered to be complete.

[0074] (6) After decalcification, rinse under running water for 24 hours. After the decalcification solution in the tissue is completely rinsed, fix it again with tissue fixative for 24 hours. After rinsing under running water for 3 hours, proceed with subsequent embedding.

[0075] 1.2.7 Micro computed tomography (Micro CT) of alveolar bone

[0076] 1.2.7.1 Analytical methods

[0077] The samples were placed in Micro CT and scanned for each group of dentate jaw specimens. The Micro CT scanning parameters used in this experiment were: scanning energy intensity of 55 KVp, 200 μA; filter of 0.5 Al; CT value corrected with 1200 mg HA / ccm; image matrix: 3072 × 3072; FOV (field of view) of 20 mm; resolution: 6.6 μm; sample time (exposure time) of 250 ms. After scanning, the Micro CT files were converted into Digital Imaging and Communications in Medicine (DICOM) files, and the images were reconstructed using NRecon (SkyScan, 2020, version 2.0) software and data browser (SkyScan, version 1.4.4) according to three planes (transverse, longitudinal, and sagittal). The distance from the enamel-cemental junction (CEJ) to the alveolarbone crest (ABC) was measured CEJ-ABC. Three sites were selected for measurement in each sample, and the mean of the three distances was defined as the representative value for each rat.

[0078] 1.2.7.2 Region of interest (ROI) parameter analysis

[0079] After selecting the ROI, the microstructural parameters of the alveolar bone were measured. The ROI area was selected based on the following standardized dimensions and anatomical landmarks:

[0080] ① The line 0.5 mm from the top of the M1 CEJ was used as the limiting line in the coronal plane and extended vertically to 1 mm from the apex;

[0081] ② A 3 mm line starting from the mesial CEJ of M1;

[0082] ③ A 2.5mm line including the buccal-palatal side. Microstructural parameters were measured using NRecon software, including: bone mineral density (BMD), tissue volume (TV), bone volume (BV). BV / TV is the bone volume fraction, which is the relative bone volume or bone volume fraction (BV / TV%), trabecular number (Tb.N), which refers to the number of intersections between bone tissue and non-bone tissue within a certain length (1 / mm). Trabecular separation / spacing (Tb.Sp), refers to the average width of the medullary cavity between trabeculae (μm), and trabecular thickness (Tb.Th), refers to the average thickness of trabeculae (μm).

[0083] 1.2.8 Histopathological analysis

[0084] 1.2.8.1 Paraffin section preparation process:

[0085] (1) Tissue embedding: The specimens stored in 75% ethanol after decalcification are sequentially immersed in 95% ethanol liquid I, II and anhydrous ethanol for 40 minutes each for step-by-step dehydration; after dehydration, they are sequentially immersed in xylene liquid I and II for 40 minutes each for tissue transparency; after transparency treatment, they are sequentially immersed in wax liquid I and II. The wax-immersed tissue is embedded in a tissue embedding machine;

[0086] (2) Sectioning: The embedded tissue blocks were quickly frozen at -20°C, and then the slice thickness of the slicer was adjusted to make 5 μm continuous tissue sections along the sagittal plane. The sections were flattened in 40°C water and placed on anti-shedding slides.

[0087] (3) Baking: Place each group of slices in a 60°C oven and bake for 30 min;

[0088] (4) Dewaxing to water: Soak the sections in three cylinders of xylene dewaxing solution I, II, and III for 10 min each until no paraffin remains on the tissue, and then soak them in 100%, 85%, and 70% ethanol for 10 min each;

[0089] (5) Water washing: Each group of slices is slowly rinsed under running water, while avoiding direct water flow to wash the tissue.

[0090] 1.2.8.2 HE staining of alveolar bone sections

[0091] The sections of each group that need HE staining for histological observation were stained. The specific steps are as follows:

[0092] (1) Hematoxylin staining: Soak the washed sections in hematoxylin stain for 15 min. After staining, wash off the excess stain under running tap water.

[0093] (2) Differentiation: The sections stained with hematoxylin were placed in a 1% hydrochloric acid ethanol solution for differentiation for 1-3 seconds, and then inverted in PBS buffer;

[0094] (3) Eosin staining: The differentiated sections were then placed in eosin staining solution for 4 min. After staining, the excess stain was washed off under running tap water.

[0095] (4) Dehydration: All sections were placed in 80%, 95% and 100% ethanol solutions I and II for dehydration;

[0096] (5) Transparent: Soak in xylene solution I and II for 5 min each;

[0097] (6) Sealing: After the slices are dried, use neutral gum to seal the slices. Observe the alveolar bone, periodontal ligament, gingival fibers and mucosa under an optical microscope to observe the structure and integrity of soft and hard tissues. Use the histopathological scoring criteria and evaluate under an optical microscope at ×100 and ×200 magnifications. HE staining is mainly used to evaluate inflammatory cell infiltration, alveolar bone resorption, and cementum degeneration and destruction in the tissue. The scoring criteria are as follows: 0 points: no or only discrete cell infiltration, the alveolar process and cementum are preserved; 1 point: moderate cell infiltration and some but slight alveolar process resorption and intact cementum; 2 points: aggravated cell infiltration, aggravated alveolar process degeneration, and partial destruction of cementum; 3 points: aggravated cell infiltration, complete resorption of the alveolar process, and severe destruction of cementum.

[0098] 1.2.8.3 Analysis of collagen fiber staining with picrosirius red

[0099] Collagen fibers are the most widely distributed and abundant fibers in connective tissues. They are widely distributed in various tissues. Type I collagen fibers are mainly distributed in bones, skin, and tendons; Type II collagen fibers are mainly cartilage collagen; Type III collagen fibers are mainly in embryonic tissues, gastrointestinal tract, etc.; Type IV collagen fibers are mainly in the basement membrane. Sirius red and its contrast dye are both strong acid dyes, which are easy to combine with the basic groups in collagen molecules and adsorb firmly. Under polarized light microscope observation, type I collagen fibers are orange-yellow or red, and type III collagen fibers are green. In this experiment, the maxillary bone sections of each group were stained with picrosirius red to analyze the collagen fiber structure. The specific steps are as follows: (1) Dewaxing and hydration: the steps are the same as HE staining; (2) Prepare iron hematoxylin staining solution before use, drip it for 5-10 minutes, wash with distilled water for 10-20 seconds to wash away the excess staining solution, and then wash with tap water for 5-10 minutes; (3) Drip the picrosirius red staining solution for 15-30 minutes. The staining time for tissues that are easy to color can be controlled within 5-10 minutes; (4) Rinse with running water to remove the staining solution on the surface of the section; (5) Differentiation, dehydration, transparency and sealing: the steps are the same as HE staining. Then, the staining of gingival tissue collagen fibers was analyzed under a polarizing microscope. In the staining results, collagen fibers were red, nuclei were green, and other structures were yellow. Images were taken under a polarizing optical microscope.

[0100] 1.2.8.4 Tartrate-resistant acidphosphatase (Trap) staining Trap is mainly divided into two categories: one is tartrate-resistant acid phosphatase and the other is fluoride-resistant acid phosphatase. Trap is a glycosylated metalloproteinase that is highly expressed in osteoclasts and chondrocytes and is considered to be the only indicator of osteoclast activity. In order to identify osteoclasts in alveolar bone, the activity of tartrate-resistant acid phosphatase was detected using the Trap kit. The sections of each group that needed Trap staining to identify osteoclasts were stained. The specific steps were as follows: (1) Dewaxing and hydration: the steps were the same as HE staining; (2) Deionized water was preheated to 37°C and the water temperature was checked again before use; (3) The fixative was placed at room temperature, and the sections were immersed in the fixative for 30 seconds and then thoroughly washed with deionized water. Do not let the sections dry out too much; wash once with PBS (37°C); (4) Prepare a 1.5mL EP tube, add fast garnet base solution and 0.5mL nitrite solution, gently invert up and down to mix for 30 seconds, and let stand at room temperature for 2 minutes. The two are mixed to form the solution in step (4); (5) Add the following liquids to a 100 mL beaker in order: 45 mL 37°C deionized water; 1 mL of the liquid in step (4); 0.5 mL naphthol AS-BI phosphate solution; 2 mL acetate solution; 1 mL tartrate solution; finally add the tartrate solution and protect from light; (6) Pour the dye solution prepared in step 5 into the dye vat and heat it to 37°C in a water bath; the temperature must be maintained at 37°C before use; (7) Place the slices in the dye vat and incubate in a 37°C water bath in the dark for 1 hour, and thoroughly wash the slides with deionized water; (8) Restain in hematoxylin solution for 2 minutes; Rinse with tap water for a few minutes until blue nuclei appear; Dry naturally and observe under a microscope. Osteoclast count: Under an optical microscope, positive cells appear purple-red and nuclei appear blue. Trap-positive cells with 3 or more nuclei located on the bone surface are defined as mature osteoclasts. The top area of ​​the interradicular bone septum of the first molar was selected as the counting area. Six high-power (×100) fields of view were selected from each section. The positive cells were counted using a single-blind method and the mean was calculated.

[0101] 1.2.8.5 Masson staining

[0102] The staining principle of this method is related to the size of anionic dye molecules and the penetration of tissues. The size of molecules is reflected by molecular weight. Small molecular weight can easily penetrate dense and low permeability tissues, while large molecular weight can only enter loose and high permeability tissues. However, the molecular weight of light green or aniline blue is very large, so after Masson staining, muscle fibers appear red and collagen fibers appear green or blue. It is mainly used to distinguish collagen fibers from muscle fibers. The specific steps are as follows: (1) After dewaxing, the sections were treated with 0.5% iodine alcohol for 10 minutes; (2) rinsed with water and treated with 5% sodium thiosulfate for 5 minutes; (3) rinsed with running water for 10 minutes; (4) stained with Weiger's iron hematoxylin for 5-10 minutes and rinsed with running water; (5) differentiated with 1% hydrochloric acid alcohol and rinsed with running water for several minutes; (6) stained with Ponceau acid fuchsin solution for 5-10 minutes and rinsed with distilled water; (6) treated with 1% phosphomolybdic acid aqueous solution for about 5 minutes, without washing with water, and directly counterstained with aniline blue solution or green solution for 5 minutes; (7) treated with 1% glacial acetic acid for 1 minute; (8) dehydrated with 95% alcohol for multiple times; (9) dehydrated with anhydrous alcohol, transparentized with xylene, and sealed with neutral gum.

[0103] 1.2.9 Immunohistochemical staining analysis

[0104] Immunohistochemical staining is the application of antigen-antibody reaction, that is, the principle of specific binding between antigen and antibody. Through chemical reaction, the color developer of the labeled antibody is used to develop color to determine the antigen in the tissue, and to conduct localization, qualitative and quantitative research on it. This is called immunohistochemistry (IHC). Each group of tissue sections that need to be analyzed by immunohistochemistry are stained. The specific steps are as follows: (1) Dewaxing and hydration: The steps are the same as HE staining, and each group of sections is grouped and labeled according to the target protein; (2) Inactivation of endogenous peroxidase: Add endogenous peroxidase blocking solution (3% H2O2) to the sliced ​​tissue, incubate at room temperature for 15 minutes, rinse with distilled water 3 times, 5 minutes each time, and then rinse with PBS buffer 3 times, 5 minutes each time; (3) Antigen repair: Place the slice in a beaker filled with citrate buffer and incubate at 95-100℃ Incubate at 37°C for 10 min, leave at room temperature until the liquid cools naturally, remove the slices, rinse with distilled water three times for 5 min each time, and then rinse with PBS buffer three times for 5 min each time; (4) Nonspecific antigen blocking (blocking nonspecific binding sites): use an immunohistochemistry pen to draw a circle around the tissue to prevent the reagent from flowing out; add 5% BSA, place the slices in a light-proof humidified box, and incubate at 37°C for 30 min; (5) Primary antibody incubation: shake off the blocking solution, add the prepared primary antibody, and quickly place the slices after the primary antibody is added in a light-proof humidified box and incubate at 4°C overnight. No more than 16 hours; after the incubation is completed, remove the incubation box, let it stand at room temperature for 40-60 minutes, and then wash it twice with PBS buffer, 5 minutes each time; (6) Secondary antibody incubation: add HRP-labeled secondary antibody to the surface of the slice tissue, place the slice in a light-proof humid box, incubate at 37°C for 30-60 minutes, and then wash it twice with PBS buffer, 5 minutes each time; (7) DAB color development: add DAB color development solution to the surface of the slice tissue for color development; observe under a microscope, control the color development time (1-2 minutes), and wait until a brown reaction appears. Stop the reaction, rinse with PBS buffer 3 times, 2 minutes each time (DAB colorimetric solution preparation: 1mL B solution + 1 drop of A solution mixed evenly); (8) Hematoxylin counterstaining: add a drop of hematoxylin staining solution on the surface of the sliced ​​tissue, counterstain for 1-2 minutes, and rinse the slide with tap water for 10 minutes; (9) Dehydration and transparency: dehydrate in 95%, 95%, 100%, and 100% ethanol, respectively, soak in each tank for 5 minutes, and then soak in xylene transparent solution I, II, and III, respectively, for 2 minutes in each tank; (10) Sealing: The steps are the same as HE staining. After staining, use an optical microscope to observe the immunohistochemical staining results of periodontal tissues and collect images. Randomly select the area of ​​interest under a ×100 optical microscope, and randomly select 6 non-repeated fields of view at ×200. Use Image Pro Plus 6.0 to analyze the staining optical density values ​​of the above 6 fields of view.

[0105] 1.2.10 RT-qPCR detection of rat gingival tissue

[0106] Sangon Biotechnology (Shanghai) Co., Ltd. was responsible for primer design and synthesis. The primer sequences are shown in Table 1.

[0107] Table 1 Primer sequences

[0108] Table I Primers sequence

[0109]

[0110] 1.2.10.1 Preparation of primer solution

[0111] Since Oligo DNA is in the form of thin dry powder and is very light and will adhere to the tube wall, after receiving the primer, it should be centrifuged at 3000-4000rpm for 30s-60s before opening the centrifuge tube cap to prevent the primer dry powder from being lost when opening the cap. Add an appropriate amount of TE buffer and prepare the primer into a primer working solution with a concentration of 10μM. Vortex the primer to mix thoroughly. Aliquot in 20μL volume, mark the name and time, and freeze at -20℃ for subsequent experiments.

[0112] 1.2.10.2 Extraction of total RNA from gingiva and alveolar bone using Trizol

[0113] (1) Pre-cool the PBS buffer in a 4°C refrigerator. The RNA extraction steps need to be performed in a biosafety cabinet on ice. (2) Take out the gingiva (3 samples per group) from a -80°C refrigerator and thaw on ice. Gently wash with cold PBS buffer and take tissue blocks of similar size (30-50 mg / sample) and put them into a tissue homogenizer. (3) Add 1 mL of Trizol to the homogenizer and grind and stir quickly to fully mix the tissue, cells and Trizol until they become a uniform paste. (4) Pour the homogenate into a pre-labeled RNAase-free EP tube and add 200 μL of chloroform. Add chloroform quickly and vortex to mix thoroughly. Shake gently for 15 seconds. (5) Let stand at room temperature for 10 minutes. After standing, centrifuge the tissue homogenate at 4°C, 12,000 rpm for 15 minutes. At this time, the liquid is divided into three layers (Trizol at the bottom, turbid proteins and cell debris in the middle, and chloroform in which RNA is dissolved in the top clear liquid); (6) prepare a new set of EP tubes, add 500 μL of isopropanol to each tube, and then carefully collect 500 μL of supernatant from the above centrifuged EP tubes (avoid inhaling turbid proteins and cell debris), ensuring that the volume ratio of isopropanol to supernatant is 1:1, invert the EP tube to mix, and let it stand at room temperature for 10 minutes, or in a -20°C refrigerator for 1 hour; (7) then centrifuge at 4°C 12000 rpm for 10 minutes, discard the supernatant, and add 1 mL of the same mixture as above. Use 75% ethanol prepared with anhydrous ethanol and DEPC water in a ratio of 3:1 to fully dissolve the RNA; (8) Then centrifuge at 4℃ and 7500rpm for 5min, discard the supernatant, repeat this process once, and use a 10μL micropipette to remove the residual ethanol; (9) Finally, turn the EP tube upside down on absorbent paper to dry. When the white precipitate becomes transparent or no obvious water droplets are seen in the tube, add an appropriate amount of DEPC water to dissolve the precipitate (the amount of DEPC water needs to be added according to the amount of RNA, generally 20μL is enough), and use a pipette to blow repeatedly until the precipitate completely disappears. At this point, the RNA extraction of animal tissues is completed. The obtained RNA solution is stored in a -80℃ refrigerator.

[0114] 1.2.10.3 RNA quantification

[0115] (1) Detect the above RNA concentration using Nanodrop2000. Select the "Nucleic Acid" detection module, and select "RNA" for the sample type. Then, clean and detect the platform using DEPC water. Next, add 1 μL of DEPC water to the detection platform and click "Blank" for blank correction. (2) After calibration, zeroing, and cleaning, add 1 μL of each group of samples in sequence for RNA concentration detection. Analyze the detection results and calculate the reaction system. Ensure that A260 / A280 is in the range of 1.8 - 2.2. After measurement, aliquot the RNA, label information such as name, volume, and date, and store it at -80 °C in the refrigerator for future use. Note: A260 / A280 < 1.8 indicates that the RNA is contaminated with protein; 1.8 < A260 / A280 < 2.2 indicates normal RNA; A260 / A280 > 2.2 indicates that the RNA has undergone hydrolysis.

[0116] 1.2.10.4 Synthesis of cDNA template and reverse transcription

[0117] (1) Thaw the RNA template and reverse transcription kit on ice in advance. Centrifuge briefly and then shake to mix the reagents thoroughly. (2) RNA cannot be used as a template for PCR reactions. It must be reverse transcribed into cDNA. This experiment uses Takara's PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, RR047A). We will perform reverse transcription experiments according to the instructions. Because the reverse transcription process is complicated, different reaction solutions need to be packaged and stored before the experiment to avoid the weakening of enzyme efficiency caused by repeated freezing and thawing, thereby reducing errors during the experiment. Prepare the reverse transcription system in a pre-cooled RNAase-free EP tube. (3) 5×gDNA EraserBuffer and gDNA Eraser need to be centrifuged briefly before use to mix evenly. After centrifuging and mixing the mixture, transfer it to the PCR instrument on an ice box and set it to 42℃, 2min, 4℃, ∞ for operation. The total volume is 10μL. Remove possible DNA contamination during the experiment. (4) Prepare the reverse transcription reaction system (reverse transcription must be completed within 25 minutes): The entire preparation process needs to be carried out on ice. In order to ensure the accuracy of the reaction system preparation, when performing each reaction, the MasterMix should be prepared according to the number of reactions + 2μL, and then divided into 10μL portions, and reverse transcription should be performed immediately after gentle mixing. (5) 5× Primescript Buffer 2 and Primescript RT Enzyme Mix1 need to be centrifuged before use. Before reverse transcription, each reaction solution needs to be fully mixed and then transferred from the ice box to the PCR instrument. The reaction is set to 37℃, 15min, 85℃, 5s, and then 4℃, ∞ in sequence. In reverse transcription, the amount of RT PrimerMix used in the TB Green qPCR method is 1μL. The amount of the obtained reaction solution added to the Real Time PCR reaction system in the next step should not exceed 1 / 10 of the total volume (V / V).

[0118] 1.2.10.5 Real-time fluorescence quantitative PCR

[0119] The obtained cDNA was diluted to twice the original volume and used as a template for subsequent RT-PCR amplification using Takara's TB Premix Ex TaqTMⅡ (TaKaRa, RR820A) fluorescent quantitative PCR reaction kit. Before using the kit, turn it upside down and gently mix it. At the same time, prevent bubbles from forming to avoid insufficient reaction due to poor mixing. Vortexing cannot be used in this process. TB Green Premix Ex Taq II stored at -20°C may produce white or light yellow precipitate, which can be slowly dissolved at room temperature and placed away from light. Mix thoroughly to dissolve all precipitates. When preparing the reaction system, all reagents should be placed on ice. The fluorescent dye TB Green should be protected from strong light. Use non-contaminated gun tips, Microtubes, etc. when preparing and packaging the reaction system to avoid contamination. (1) Prepare the PCR reaction solution according to the instructions and keep it on ice during the preparation process. Taking into account the loss during aspiration, the volume of the prepared premix should be 10-15% more than the total volume; (2) After preparing the reaction solution, use a fluorescent quantitative PCR instrument to perform the amplification reaction, using the two-step PCR amplification standard procedure: pre-denaturation cycle number: 1, 95°C 30s; PCR reaction cycle number: 40, 95°C 5s, 60°C 34s; (3) Analyze the data.

[0120] 1.2.11ELISA detection of serum inflammatory factor concentrations

[0121] Before the assay, take one aliquot of frozen serum samples from each group (avoid repeated freezing and thawing) and centrifuge at 4°C 1000g for 15 min. Then determine the concentrations of Il6, Mmp9, Ilβ and TNFα in serum and gingival crevicular fluid according to the instructions of the kit. Before the experiment, place the reagents in the kit at room temperature for 30 min, equilibrate at room temperature, and prepare them according to the instructions for use. (1) Preparation of standard: According to the instructions, take out one standard and centrifuge it at 6000-10000rpm for 30s. Dissolve it with 1mL of sample diluent, and blow repeatedly with the tip of the gun at the bottom of the cryopreservation tube to help dissolve it. After fully mixing, obtain standard S7. After dissolution, perform gradient dilution according to the standard dilution method in the instructions to obtain standards of corresponding concentrations (S6-S1), and mix them by gently pipetting. S0 is the sample diluent; (2) Preparation of washing working solution: dilute the concentrated washing solution with deionized water at a ratio of 1:25; prepare it before use; the concentrated washing solution will precipitate salts when stored at low temperature, and it can be heated in a water bath to help dissolve it when diluted; (3) Loading: take out the pre-coated ELISA plate, and set up standard wells and test sample wells respectively. Carefully add the standard and the sample to be tested (100 μL / well) to the well plate, shake gently to mix, cover the plate sticker, and incubate at 37°C for 2 hours. After the incubation, take out the experimental ELISA plate, remove the sealing film, shake off the sample and standard solution, and spin dry on lint-free absorbent paper and tap the plate 5-10 times without washing; (4) Dilution of biotinylated antibody: dilute the biotinylated antibody solution with biotinylated antibody diluent at a ratio of 1:100. For example, add 10 μL of biotinylated antibody to 990 μL of biotinylated antibody diluent, mix gently, and prepare within 10 minutes before use; (5) Add biotinylated antibody: add 100 μL / well of diluted biotinylated antibody solution to each well, cover with a new plate sticker, and incubate at 37°C for 1 hour; after incubation, discard the liquid in the well, spin dry, wash the plate 3 times, soak for 2 minutes each time, 200 μL / well, and spin dry; (6) Prepare horseradish peroxidase-labeled avidin working solution: dilute horseradish peroxidase-labeled avidin with its diluent at a ratio of 1:100. (7) Add horseradish peroxidase-labeled avidin: Add 100 μL of diluted horseradish peroxidase-labeled avidin working solution to the washed wells, cover with a new plate sticker, and incubate at 37°C for 1 hour; after incubation, discard the liquid in the wells, spin dry, wash the plate 5 times, soak for 2 minutes each time, 200 μL / well, spin dry; (8) Color development: Add 90 μL of substrate solution to each well of the washed plate in sequence, and color develop at 37°C in the dark for 15-30 minutes; (9) Stop the reaction: Add 50 μL of stop solution to each well in sequence to stop; (10) Read the value: Within 5 minutes after termination, use an enzyme reader to measure the optical density of each well in sequence at a wavelength of 450 nm. Save the data and analyze the concentrations of Il6, Mmp9, Ilβ and TNFα in serum.

[0122] 1.3 Statistical analysis

[0123] The data obtained from the experiment are expressed as mean ± SD, and the mean is the average of three independent repeated experiments. SPSS26.0 (SPSS Inc. USA) software was used to test the normality of quantitative data. The data in this study were all in accordance with normal distribution. The variance homogeneity test was performed. If the variance was homogeneous, one-way ANOVA was used. If the differences between the groups were statistically significant, the LSD method was used to test the differences between the groups; if the variance was unequal, the Kruskal-Wallis rank sum test in the nonparametric test was used for pairwise comparison. The test level α = 0.05. Semi-quantitative analysis of immunohistochemistry was performed using Image J 6.0 software. Bar graphs and line graphs were drawn using GraphadPrism 9.0 (graphpad, LaJolla, CA, USA) software.

[0124] 2 Results

[0125] 2.1 Basic indicators

[0126] 2.1.1 Weight

[0127] The body weight of rats in the BG group showed a steady growth trend throughout the experimental period, without sudden decrease or increase, which was consistent with the normal growth pattern of rats; in contrast, the body weight of the remaining modeling experimental groups first decreased and then increased after the modeling was completed. This was because periodontal discomfort after modeling affected the rats' eating. As time passed, after the acute phase of inflammation, the discomfort decreased, and the rats' eating recovered. The drug group has the effect of controlling inflammation. The inflammation control time of rats was short, and the eating recovery time was shorter than that of the non-drug intervention group. The body weight in the later stage was higher than that of the PG and GG groups. Due to the use of glycerol as a solvent, some rats struggled during the medication process, and accidentally ingested glycerol, causing mild diarrhea. The increase in solid weight would fluctuate, but it would not affect the overall trend. For details of the results, see Figure 1 shown.

[0128] 2.1.2GBI and PPD

[0129] During the experiment, the GBI and PPD of rats were recorded daily, and the data were summarized to draw line graphs and bar graphs to reflect the severity of periodontitis. We found that the scores of clinical indexes of periodontitis in the PG and GG groups were similar but significantly higher than those in the other groups (P<0.05), indicating that glycerol as a solvent had no effect on treating experimental periodontitis in rats. In addition, the medium / high concentration groups of mulberry leaf extract and the positive drug minocycline hydrochloride could alleviate the clinical symptoms of experimental periodontitis, among which the MH group performed best; the ML group also had a certain effect on reducing the clinical indexes of experimental periodontitis, but it was not significant. According to the line graph of the changes in GBI of each group during the experiment, we found that: in the early stage of ligation, the clinical score showed an increasing trend, and in the late stage of ligation, the scores of each group subject to drug intervention showed a decreasing trend, and the difference with the PG group gradually increased, and the gingival bleeding index decreased. Similarly, after ligation, the PPD of each group first increased, then decreased, and then became stable. At the end of the experiment: the periodontal pockets in the HM and PG groups were the shallowest, and the difference with the BG group was the smallest, but the difference with the PG and GG groups was significant (P<0.05). The TM values ​​of the other groups at the end of the experiment were also significantly different from those of the EP and GG groups (P<0.05). Figure 3 shown.

[0130] 2.2 Imaging indicators

[0131] 2.2.1 Distance from cementoenamel junction to alveolar crest (CEJ-ABC)

[0132] After the experiment, the rats in each group were scanned by Micro CT and three-dimensionally reconstructed. The distance between the cementoenamel junction and the alveolar crest was measured at the mesial, central and distal sites of the maxillary first molar. The measurement results showed that after 2 weeks of the experiment, the CEJ-ABC of the PG group was much larger than that of the BG group, MH group and PC group (P<0.05), and the distance values ​​of the MH group and PC group were closest to the BG group. Analysis of imaging results showed that: sagittal plane: the height of the alveolar crest was reduced in the PG and GG groups; obvious bone defects appeared in the distal and furcation areas, and the alveolar bone was porous; while the alveolar bone loss was less in the drug intervention group, and the bone in the MH and PC groups was more homogeneous and dense, similar to the blank control group; the coronal plane and sagittal plane results were basically consistent; horizontal plane: the PG and GG groups had osteoporosis, reduced density, and enhanced trabecular radiolucency; although the drug intervention group had different degrees of low-density radiolucency, the area was not as obvious as that of the PG group; and the alveolar bone structure of the BG group was clear and the bone was uniform. Figure 4 shown.

[0133] 2.2.2 Alveolar bone resorption

[0134] TRI / 3D-BON software was used to measure the alveolar bone structure parameters and analyze the parameters. BMD: Compared with BG, the bone density of PG and GG groups was significantly lower (P<0.05); compared with PG and GG groups, the bone density of MC, NH, and MH groups was higher than that of PG group, and the bone density of MH group was similar to that of BG group. Figure 5 BV / TV (%): Compared with BG, the BV / TV values ​​of PG and GG groups were significantly lower (P<0.05); compared with PG and GG groups, the BV / TV of the drug intervention group was higher than that of the PG group, and the BV / TV of the MH group was similar to that of the BG group. Figure 6 (C) in the figure. Trabecular thickness Tb.Th: Compared with the BG group, the trabecular thickness of the PG and GG groups was significantly reduced (P<0.05); compared with the PG and GG groups, the trabecular thickness of the drug intervention groups was higher than that of the PG group, and the Tb.Th value of the MH group was close to that of the BG group. Trabecular number Tb.N: Compared with the BG group, the trabecular number of the PG and GG groups was significantly reduced (P<0.05); compared with the PG and GG groups, the number of trabeculae in the drug intervention groups was more than that in the EP group. Trabecular separation Tb.Sp: Compared with the BG group, the trabecular separation of the PG and GG groups was significantly increased (P<0.05); compared with the PG and GG groups, the trabecular separation of the drug treatment groups was reduced, and the trabecular separation of the MH group was the lowest. The results are shown in Figure 5 The above results all indicate that a certain concentration of mulberry leaf extract can effectively reduce the loss of periodontal supporting tissue, while increasing bone density, trabecular thickness and strength, and the number of trabecular bone, thereby improving the quality of alveolar bone.

[0135] 2.3 Histopathological analysis

[0136] 2.3.1 HE staining

[0137] HE staining results showed that after 14 days of the experiment, typical inflammatory lesions of periodontal tissues appeared around the first molars in the PG and GG groups, which were consistent with the imaging findings. The gingival papilla was absorbed below the cementoenamel junction, the height of the alveolar ridge was reduced, and deep periodontal pockets appeared in combination with the root proliferation of the epithelium. The gingival fibers were partially broken and disordered, and there were a large number of inflammatory cell infiltrations in the periodontal tissues, mainly neutrophils and lymphocytes. Obvious bone resorption pits were also observed in some samples. The periodontal inflammation in rats that applied the drug locally was alleviated, showing mild or moderate inflammatory cell infiltration. At the same time, the alveolar bone and gingival fiber structures were relatively intact. For details, see the results. Figure 7 According to the inflammation score of periodontal tissue, the degree of alveolar bone loss and severity of inflammation in MH, MM and PC groups were significantly lower than those in PG and GG groups (P<0.05), and the MH group had the best effect.

[0138] 2.4.2 Sirius red staining

[0139] Sirius red staining needs to be performed under a polarized light microscope. Compared with ordinary optical microscopes, the background of images collected under a polarized light microscope is black, and the collagen fiber bundles show different colors (red, yellow and green) due to different arrangements under this background. The destruction of gingival collagen fibers in each group is compared, and the results are detailed in Figure 2-7 A. The gingival collagen fiber bundles in the PG group were scattered and disordered, and some were even broken and missing to form black hollow areas. The collagen fibers in the ML group had no obvious breaks or hollows, but were arranged loosely. The HM, CG, and MM groups showed the protective ability of mulberry leaf extract on the collagen fibers in the gums, and the fibers were arranged neatly and orderly. At the same time, the semi-quantitative analysis of collagen fibers was performed by calculating the ratio of stained fibers to the measured area. The results showed that the ratio of stained fibers to the measured area was 1. Figure 8 As shown in (D) in the figure, there was no significant difference between the PG group and the GG group (P>0.05), but there was a significant difference between the HM group and the PG group and the GG group (P<0.05).

[0140] 2.4.3 Masson staining

[0141] Masson staining was used to observe the periodontal collagen arrangement and repair. The tissue staining morphology of the HM and PC groups was similar to that of the BG group, showing more orderly and denser collagen deposition (blue reticular fiber structure) than the PG group, and closely attached to the tooth root; while the collagen fiber bundles in the PG group were broken and the collagen deposition was disordered. This experiment shows that mulberry leaf extract can effectively promote the remodeling and deposition of periodontal collagen, which is beneficial to the tissue repair of periodontitis. The results are shown in Figure 8 As shown in (B) in the figure. Semi-quantitative analysis of collagen fibers was performed by calculating the ratio of stained fibers to the measured area. The results are shown in Figure 8 As shown in (E) in the figure, there was no significant difference between the PG group and the GG group (P>0.05), but there was a significant difference between the HM group and the PG group and the GG group (P<0.05).

[0142] 2.4.4 Trap staining

[0143] Trap-positive cells are distributed in the alveolar bone. The cytoplasm of mature osteoclasts is purple-red, the nucleus is blue, and each cell has at least three or more nuclei. We selected three areas, the mesial and distal adjacent areas and the root bifurcation, to count osteoclasts and perform statistical analysis. Similar to the HE staining results, the number of Trap-positive cells in the PG and GG groups was much higher than that in the BG group (P<0.05). The number of positive cells in the drug intervention groups decreased to varying degrees. Among the experimental groups, the HM and CGT groups had the least number of Trap-positive cells, showing excellent ability to inhibit osteoclasts (P<0.05). The results are shown in Figure 8(C)(F) in.

[0144] In summary, the above experiments have demonstrated the therapeutic effect of mulberry leaf extract on rat periodontitis, and the pathogenesis of the rat ligature-induced model is similar to that of human periodontitis, indicating that mulberry leaf extract has the potential to be used as a periodontal therapeutic drug. The mulberry leaf extract group in the scheme of the present invention can significantly downregulate the expression of mRNA related to inflammation and HIF pathway, and upregulate the expression of mRNA related to osteogenesis. It is proved that the local application of mulberry leaf extract has a therapeutic effect on experimental periodontitis in rats, and mulberry leaf extract can be used as a potential periodontal preventive and therapeutic drug.

[0145] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for in vitro proliferation of bone marrow mesenchymal stem cells, characterized in that: The method comprises the following steps: S1. preparing rat bone marrow mesenchymal stem cells, culturing them at a constant temperature of 36°C to 37°C under a 5% CO2 condition, and then passage them; S2. Take the third generation of rat bone marrow mesenchymal stem cells and adjust the cell concentration to 1×10 7 After the cells were grown in the basal medium, mulberry leaf extract was added to the basal medium. S3, then add CCK-8 reagent and incubate in a CO2 incubator in the dark for 3 hours to detect the proliferation of rat bone marrow mesenchymal stem cells.

2. The in vitro proliferation method of bone marrow mesenchymal stem cells according to claim 1, characterized in that: The amount of mulberry leaf extract added in step S2 is 10 mg / L-150 mg / L.

3. The in vitro proliferation method of bone marrow mesenchymal stem cells according to claim 2, characterized in that: The amount of the mulberry leaf extract is 10 mg / L.

4. The in vitro proliferation method of bone marrow mesenchymal stem cells according to claim 1, characterized in that: The concentration of the mulberry leaf extract is 1-100 μg / mL.

5. Application of mulberry leaf extract in the proliferation of bone marrow mesenchymal stem cells.

6. The in vitro proliferation method of bone marrow mesenchymal stem cells according to claim 4, characterized in that: The method is used in preparing medicine for preventing and treating periodontitis.

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