CN113841658A - A kind of rapid establishment method of periodontitis model and treatment drug - Google Patents
A kind of rapid establishment method of periodontitis model and treatment drug Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
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- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/02—Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
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Abstract
本发明公开了一种牙周炎模型的快速建立方法及治疗药物，通过大鼠上颌双侧第二磨牙并配合5天高糖饮食成功得到单纯性大鼠牙周炎模型。本发明建立大鼠牙周炎模型后，将中性三七多糖应用于本模型，为三七资源的综合利用提供参考依据，给予中性三七多糖治疗后，大鼠炎症得到缓解，显著抑制了牙槽骨丢失，极大的保护了牙龈出血，本发明增加了中性三七多糖用于牙周炎治疗的新用途，为中性三七多糖治疗牙周炎的生物研究。
The invention discloses a method for rapidly establishing a periodontitis model and a therapeutic drug. The simple rat periodontitis model is successfully obtained by combining the rat maxillary bilateral second molars and a 5-day high-sugar diet. After the rat periodontitis model is established in the present invention, the neutral Panax notoginseng polysaccharide is applied to the model to provide reference for the comprehensive utilization of Panax notoginseng resources. The alveolar bone loss is significantly inhibited, and gingival bleeding is greatly protected. The present invention increases the new use of the neutral Panax notoginseng polysaccharide for the treatment of periodontitis, which is a biological research on the treatment of periodontitis with the neutral Panax notoginseng polysaccharide. .
Description
Technical Field
The invention relates to the application field of pseudo-ginseng natural products, in particular to a method for quickly establishing a periodontitis model and a therapeutic drug.
Background
Periodontitis is a common chronic infectious disease, is very common and prevalent in oral diseases, is characterized by connective tissue attachment and alveolar bone loss, is often accompanied by symptoms such as tooth loosening and gingival bleeding, and is a main reason for adult tooth loss, and nowadays, many studies have proved that the initiation factor of periodontitis is the accumulation of bacterial plaque biofilm around gingiva, which in turn triggers a host immune response mediated by a series of inflammatory cytokines. In the whole process of periodontitis pathology, the IL-1 beta level is high, which can promote the proliferation and activation of osteoclast and participate in the destruction of periodontitis tissue structures and the loosening and falling of teeth. The rat is a common animal for establishing the periodontitis model and has the advantages of economy and high efficiency, and common methods for establishing the periodontitis model comprise a bacterium coating method, an LSP method, a ligation method matched with a long-term high-sugar diet method and the like. Compared with steel wire ligation, other methods have the defects of complicated steps, need of re-fixing the silk threads and the like. Researchers successfully obtain a rat periodontitis model by ligating orthodontic 2-0 steel wires and matching with a 20% high-sugar diet induction model for four weeks, but long-term high-sugar diet can induce metabolic diseases of an organism and can also damage the immune system of the organism, so that the researches on the simple periodontitis model are not facilitated.
Pseudo-ginseng (Panax notoginseng is a traditional famous traditional Chinese medicinal material in China, has wide application and contains various active substances, so far, the research on pseudo-ginseng is mainly focused on pseudo-ginseng saponin, but the research on pseudo-ginseng polysaccharide is less, pseudo-ginseng polysaccharide is one of main pharmacodynamic active substances of pseudo-ginseng, and the research proves that the pseudo-ginseng polysaccharide has in-vitro immunostimulating activity, immunity regulating and anti-tumor activity, nerve protecting function, strong free radical scavenging activity capability and anti-inflammatory and bone injury repairing effects.ZL 201710459344 describes the extraction and purification method of pseudo-ginseng neutral polysaccharide and the effect of promoting the in-vitro proliferation of human periodontal ligament stem cells and mouse osteoblasts, but the treatment effect of the neutral pseudo-ginseng polysaccharide on rat periodontitis is not reported yet, so that a method for quickly establishing a periodontitis model and a treatment medicine are needed.
Disclosure of Invention
The invention aims to provide a simple and easy method for establishing a periodontitis model with an ideal effect, and a method for quickly establishing the periodontitis model by applying neutral pseudo-ginseng polysaccharide to the model and a treatment medicament.
A method for establishing a periodontitis model comprises ligating second upper jaw molars of a rat by using 2-0 orthodontic steel wires and matching with 5-day high-sugar diet to successfully obtain the rat periodontitis model.
A medicine for treating periodontitis model contains neutral Notoginseng radix polysaccharide 85%.
Further, neutral notoginseng polysaccharide with the content of about 85 percent is used for the model in a gastric lavage mode, the administration dosage is respectively 200, 400 and 800mg/kg, the administration is carried out once a day, and the administration lasts for 28 days.
The periodontitis model is successfully obtained by applying the method, and the periodontal inflammation is greatly relieved after the neutral pseudo-ginseng polysaccharide is applied to the model: a decrease in serum IL-1 β levels; the distance from the enamel nature boundary to the crest of the alveolar ridge is obviously reduced; the percentage of bone resorption area in the bifurcation area is significantly reduced; the gingival bleeding condition is obviously improved; the loosening of the molar teeth is relieved.
The invention has the beneficial effects that:
after a rat periodontitis model is established, neutral pseudo-ginseng polysaccharide is applied to the model, a reference basis is provided for comprehensive utilization of pseudo-ginseng resources, inflammation of the rat is relieved after the neutral pseudo-ginseng polysaccharide is used for treatment, alveolar bone loss is remarkably inhibited, gingival bleeding is greatly protected, and in addition, the odontoseisis improved.
Drawings
FIG. 1 is a graph showing the experimental results of the effect of neutral notoginseng polysaccharides on IL-1. beta. levels in rat periodontitis model serum in example 1.
FIG. 2 is a graph of experimental results of the effect of neutral Notoginseng radix polysaccharide on distance from enamel junction to alveolar ridge crest (CEJ-ABC) in rat periodontitis model in example 1.
FIG. 3 is a graph showing the experimental results of the effect of the neutral notoginseng polysaccharides on pathological sections of the second molar root bifurcation region in the rat periodontitis model in example 1.
FIG. 4 is a graph of experimental results of the effect of neutral Notoginseng radix polysaccharide on the percent bone resorption in the bifurcation of the second molar root in the rat periodontitis model in example 1.
Detailed Description
In order to further understand the technical features of the present invention, the present invention is described in detail with reference to the specific embodiments below. The embodiments are given by way of illustration only and not by way of limitation, and any insubstantial modifications, based on the present disclosure, may be made by those skilled in the art without departing from the scope of the present disclosure.
A method for establishing a periodontitis model comprises ligating second upper jaw molars of a rat by using 2-0 orthodontic steel wires and matching with 5-day high-sugar diet to successfully obtain the rat periodontitis model.
Example 1
Establishment of rat periodontitis model and application of neutral pseudo-ginseng polysaccharide to model
(1) Model establishment and grouping: 30 SPF SD rats are selected from males, 5 weeks old and 100-140g in weight, and the weight is about 200g after 2 weeks of adaptive conventional feeding. 25 patients were randomly selected as a model group for modeling progressive periodontitis, and the remaining 5 patients were selected as a normal group (N) and were not treated. The periodontitis molding method comprises the following steps: carrying out sexual intraperitoneal injection anesthesia (40mg/kg) on a rat by using a pentobarbital sodium solution, fixing the rat in a supine position, selecting second molars on two sides of the upper jaw, stripping the gum, ligating the second molars by using a 3.0 medical orthodontic steel wire, positioning a ligation head in a gingival sulcus as much as possible, and randomly dividing a model group into a model group, a minocycline hydrochloride positive control group, a neutral pseudo-ginseng polysaccharide low (200mg/kg), medium (400mg/kg) and high (800mg/kg) dose treatment group (n is 5) after the ligation is finished. From the day of ligation, the model-building rats were fed with soft food soaked in 10% high-sugar water and 10% high-sugar water, and were continuously fed for 5 days, and from day 6 onward, they were fed conventionally. The normal group was fed in a conventional manner throughout the experiment. From the day after ligation, each group of rats was gavaged as in table 1 for 28 consecutive days.
TABLE 1 Experimental groups
As shown in figure 1, compared with the normal group of rats, the method remarkably increases the serum IL-1 beta level (P <0.01) of the rats in the model group, the morphological result shows that the alveolar bone absorption distance of the model group is remarkably higher than that of the normal group (P <0.01, see figure 2), and the experimental result shows that the rat periodontitis model can be successfully obtained by applying the method.
A medicine for treating periodontitis model contains neutral Notoginseng radix polysaccharide 85%.
(2) Reagent preparation and administration: accurately weighing neutral Notoginseng radix polysaccharide, dissolving in sterile distilled water, preparing neutral Notoginseng radix polysaccharide reagents with three concentrations of 20mg/mL, 40mg/mL and 80mg/mL respectively according to doses of 200mg/kg, 400mg/kg and 800mg/kg, and administrating by intragastric administration of 1mL polysaccharide per 100g weight of rat; weighing a proper amount of minocycline hydrochloride, dissolving the minocycline hydrochloride in sterile distilled water to ensure that the concentration of the minocycline hydrochloride is 2mg/mL, and per 100g of weight of rat, gavaging 1mL of minocycline hydrochloride reagent; the model group was intragastrically filled with the corresponding volume of sterile distilled water.
Examining each index after the administration period is over
(1) And (3) checking the cell factors: after the last administration, the rats in each group are fasted for 12 hours without water prohibition, the rats are subjected to abdominal anesthesia by using a pentobarbital sodium solution, about 4mL of abdominal aorta blood is collected by a vacuum blood collection tube, the abdominal aorta blood is kept standing for 20min at room temperature, centrifugation is carried out for 15min at 3000rpm/min at 4 ℃, serum is obtained by careful separation, split charging and storage are carried out at 20 ℃ below zero, the level of a serum inflammatory factor IL-1 beta is detected by an enzyme-linked immunosorbent assay (ELISA), and the strict operation is carried out according to the kit specification.
(2) And (3) morphological observation: each group of rats was sacrificed by removing the neck, the maxillary bones on both sides were carefully separated, the maxillary bone on the left side was peeled off tissues such as gingiva and periosteum, boiled for 5min to remove the tissues better, washed and soaked in 0.2moL/L NaOH solution for 5min to remove the residual soft tissue debris, taken out and washed again, and 1% methylene blue was stained for 5min after air drying to determine the enamel dentinal junction (CEJ). After being photographed by a mobile phone, the distance from the enamel dentin boundary at each of 3 points on the buccal and jaw sides to the crest of the alveolar ridge is measured by using image analysis software ImageJ.
(3) And (3) gum index determination: after the last administration, rats in each group were fasted and not forbidden for 12h, the rats were anesthetized with pentobarbital sodium solution in the abdominal cavity, and bilateral gingival indexes of the rats were scored with probes according to the following scoring criteria:
no bleeding occurs when 0 minute-needle is used, and the color is normal;
score 1-mild inflammation, mild edema, slight color change and no bleeding on detection;
2 points-moderate inflammation, edema, bright and red gum, bleeding probe;
score 3-severe inflammation, marked redness, edema, ulceration and tendency to spontaneous bleeding;
(4) measuring the molar looseness: after the last administration, rats in each group were fasted and kept for 12h, and were anesthetized with pentobarbital sodium solution in the abdominal cavity, and the bilateral molar loosening degree of the rats was scored with a probe, and the scoring criteria were as follows:
0 minute-molar non-loosening
1 minute-molar loss in only the buccal and lingual directions
2 minute-molar loosening in the bucco-lingual and mesial-distal directions
3 fen-molar loosening in buccal, mesial, and perpendicular (up-down) directions
(5) Histopathological analysis: after the last administration, fasting and water prohibition are carried out for 12h, each group of rats is killed by removing the neck, the upper jaw bone on the right side is taken, 4% paraformaldehyde is put into the rats and fixed for 24h at 4 ℃, then, decalcification is carried out for 40 days by using 10% EDTA at 4 ℃, dehydration is carried out by using alcohol with series concentration, paraffin embedding is carried out, tissue section is carried out, the thickness of the section is 5 mu m, HE staining is carried out, the bone absorption condition and the pathological change of gum tissue in the dental root bifurcation area are observed under a 40X electron microscope, and the proportion of the bone absorption nest to the bone area of the root bifurcation is calculated by using ImageJ software.
Effect of neutral Notoginseng radix polysaccharide on rat periodontitis model
The neutral notoginseng polysaccharide with the content of about 85 percent is applied to the model in a gastric lavage way, the administration dosage is respectively 200mg/kg, 400mg/kg and 800mg/kg, the administration is carried out once a day, and the administration lasts for 28 days.
(1) The neutral notoginseng polysaccharide can slightly reduce the IL-1 beta content of the serum of a rat with periodontitis: as shown in fig. 1, the IL-1 β content in the model group was significantly increased compared to the normal group with statistical difference (P < 0.01). Compared with the model group, the IL-1 beta content of the minocycline hydrochloride group and the neutral notoginseng polysaccharide-400 mg/kg group is reduced, which shows that the neutral notoginseng polysaccharide has the function of inhibiting the inflammation of the rat with periodontitis.
(2) The neutral notoginseng polysaccharide can obviously inhibit the absorption distance from the enamel essence boundary to the crest of the alveolar ridge of a rat with periodontitis: the morphological results are shown in fig. 2 and 3, compared with the normal group, the alveolar bone resorption distance of the model group reaches 848.86mm, and the significant difference (P <0.01) indicates that the rat periodontitis model is successful. Compared with the model group, the alveolar bone absorption degree of rats in each group is relieved, wherein the alveolar bone absorption of the neutral panax notoginseng polysaccharide-400 mg/kg group is reduced to 683.12mm, the significant difference is realized (P <0.01), and the minocycline hydrochloride group and the neutral panax notoginseng-200 mg/kg group are respectively reduced to 717.46mm and 733.1mm (P < 0.05).
(3) The neutral notoginseng polysaccharide can obviously improve the gingival bleeding condition of a rat with periodontitis: the results of the neutral notoginseng polysaccharide effect on the gum index of the periodontitis rat are shown in fig. 4 and table 2, the gum index of each group of rats is obviously increased compared with the N group (P <0.01), the gum index of each dose group of the neutral notoginseng polysaccharide is reduced compared with the PD group, and the neutral notoginseng polysaccharide-400 mg/kg group and the neutral notoginseng polysaccharide-200 mg/kg group have statistical difference (P <0.01, P < 0.5).
TABLE 2 gingival bleeding index
Note P <0.05, P <0.01 compared to normal group; compared to the model group, # P <0.05.# P <0.01.
(4) The neutral notoginseng polysaccharide can slightly inhibit the molar looseness of a rat with periodontitis: as shown in table 3, the loosening of molars of rats in each group was increased compared to the normal group, the molar index was decreased in each dose group of minocycline hydrochloride and neutral notoginseng polysaccharide compared to the model group, and the neutral notoginseng polysaccharide-400 mg/kg group showed the best effect, but there was no statistical difference.
TABLE 3 influence of neutral Notoginseng radix polysaccharide on molar loosening
(5) The neutral pseudo-ginseng polysaccharide can obviously inhibit the further development of a bone absorption pit in a bifurcation area of a molar root: HE staining results are shown in figures 3 and 4: the gingival papilla structure between the second molar and the first molar of the upper jaw of the rat in the normal group is complete, the gingival tissue is full of gaps, the connective tissue does not migrate to the root, the bone absorption pit is occasionally seen in the bifurcation area of the second molar root, and the shape is normal. The gum tissue structure of other groups is incomplete, the gum papilla disappears, ulcer necrosis with different degrees occurs, and connective tissue migrates to root direction with different degrees. Wherein, a great amount of bone absorption pits can be seen in the crotch area of the molar root of the model group, the bone absorption area reaches 18.7 percent and is obviously larger than that of the bone absorption pits of the normal group (P is 0.005), the bone absorption areas of the minocycline hydrochloride and the neutral notoginseng polysaccharide-400 mg/kg groups are respectively 9.3 percent and 9.8 percent and are obviously lower than those of the model group (P is 019, P is 0.022).
The action mechanism of the neutral notoginseng polysaccharide for treating periodontitis is completely different from that of a common medicine minocycline hydrochloride for clinically treating periodontitis. Minocycline hydrochloride primarily inhibits periodontal bacterial infection, which in turn inhibits host immune response. In the invention, the neutral notoginseng polysaccharide has obvious inhibition effect on the absorption of alveolar bone of a rat with periodontitis, which probably reduces the level of IL-1 beta in serum of the rat, further inhibits the activation and activation effect of the IL-1 beta on osteoclast to a certain extent, and finally inhibits the absorption of bone.
The periodontitis model is successfully obtained by applying the method, and the periodontal inflammation is greatly relieved after the neutral pseudo-ginseng polysaccharide is applied to the model: a decrease in serum IL-1 β levels; the distance from the enamel nature boundary to the crest of the alveolar ridge is obviously reduced; the percentage of bone resorption area in the bifurcation area is significantly reduced; the gingival bleeding condition is obviously improved; the loosening of the molar teeth is relieved.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
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