CN114557985A

CN114557985A - Fritillaria-lanning composition for treating osteoarthritis and application thereof

Info

Publication number: CN114557985A
Application number: CN202210251539.9A
Authority: CN
Inventors: 林苇舟; 蒋福升; 李美芽; 丁滨; 张春椿; 李石清; 袁强
Original assignee: Zhejiang Chinese Medicine University ZCMU
Current assignee: Zhejiang Chinese Medicine University ZCMU
Priority date: 2022-03-15
Filing date: 2022-03-15
Publication date: 2022-05-31
Anticipated expiration: 2042-03-15
Also published as: CN114557985B

Abstract

The invention discloses a fritillaria and thunberg fritillary bulb composition for treating osteoarthritis and application thereof, wherein the fritillaria and thunberg fritillary bulb composition is prepared by mixing fritillaria and thunberg yam rhizome III according to the mass ratio of 1:1-1: 3. The calanine composition disclosed by the invention can better inhibit MMP13 expression and promote COL2A1 expression, thereby inhibiting IL-1 beta-induced cartilage matrix degradation and showing stronger inhibition and protection effects on the cartilage matrix degradation of a mouse DMM model. Therefore, the combination of the caladium and the yam essence III can be used for medicines and health-care products which have main functions of preventing and treating osteoarthritis.

Description

Fritillaria-lanning composition for treating osteoarthritis and application thereof

(I) technical field

The invention relates to a fritillaria and lanning composition for treating osteoarthritis and application thereof.

(II) background of the invention

Osteoarthritis (OA) is a common chronic degenerative joint disease with pain, swelling, and movement disorders as the major symptoms. Its pathogenesis is complex and diverse, is not clearly understood, but basically accepts that the inflammatory response plays an important role in the initial stage of the disease. At present, the non-steroidal anti-inflammatory drug is a common drug for treating OA, but only has the effect of relieving joint pain, the joint degeneration is not improved, the liver and kidney functions can be influenced after long-term use, and the risk of cardiovascular events is increased. The disease is well developed in middle-aged and elderly people, the life quality of the people is seriously influenced, and meanwhile, the disease also brings huge burden to medical health and national economy. Therefore, there is an urgent need to develop more effective drugs for treating OA.

Studies have shown that an increased metabolic activity of chondrocytes and an imbalance between extracellular matrix synthesis and catabolism are one of the important factors responsible for the pathogenesis of OA. In this process, the overexpression of matrix metalloprotease, such as matrix metalloprotease 13(MMP13), is a key factor leading to the degradation of extracellular matrix, and the decrease of extracellular matrix synthesis, such as insufficient synthesis of type ii collagen α 1(COL2a1), is another important factor for the development and progression of OA. Therefore, MMP13 and COL2A1 are important target molecules for developing OA treatment medicines, and a plurality of traditional Chinese medicine single and compound medicines with good prevention and treatment effects on OA show good regulation effects on the two target molecules, such as a salvia miltiorrhiza extract, a peach kernel and knee rehabilitation pill, a qi-tonifying, blood stasis-removing and kidney-tonifying prescription and the like, and show good development and application prospects. However, the traditional Chinese medicine and the compound have the problems of unclear effective components, lack of index components, unstable curative effect and the like, so that the modern pharmaceutical technology means is necessary to be adopted for standardization.

The research shows that the independent effects of the traditional Chinese medicine monomer components of the fritillaria lanning and the dioscin III have no obvious inhibition effect on the IL-1 beta-induced degradation of the cartilage cell matrix, but the combination of the two components can obviously inhibit the degradation of the matrix; and further proved by an in vitro cartilage experiment and a mouse in vivo osteoarthritis model, the composition with the mass ratio of 1:2 can inhibit MMP13 and promote expression of COL2A1, and has strong inhibition and protection effects on IL-1 beta induced cartilage matrix degradation and medial meniscus tibial ligament detachment osteoarthritis (DMM) cartilage matrix degradation. The treatment effect and the molecular mechanism of related compounds of the fritillaria blanco, the dioscin III and the composition thereof on the aspect of osteoarthritis are not reported in documents, and the compound has obvious innovation.

Disclosure of the invention

The invention aims to provide a peiminine composition for treating osteoarthritis and application thereof, the peiminine composition can achieve the effect of preventing or treating osteoarthritis by inhibiting MMP13 expression and promoting COL2A1 expression, the peiminine and yam element III have definite components and structures and clear dosage relation, and the peiminine and yam element III have important significance for preparing OA treatment medicines with reliable quality and definite curative effect, and have wide application prospects in the aspect of treating osteoarthritis.

The technical scheme adopted by the invention is as follows:

the invention provides a fritillaria and thunberg fritillary bulb composition for treating osteoarthritis, which is prepared by mixing fritillaria and thunberg yam rhizome III according to the mass ratio of 1:1-1:3, and preferably 1: 2.

The structural formulas of the fritillaria thunbergii ningglory and the yam essence III are as follows:

the fritillaria thunbergii and the yam essence III can be prepared by the existing known method.

The invention also provides application of the peiminine composition in preparation of a medicine for treating osteoarthritis, wherein the medicine is a medicine for inhibiting expression of matrix metalloproteinase 13(MMP13) and promoting expression of type II collagen alpha 1(COL2A 1). More preferably, the medicament is a medicament for treating articular cartilage degradation and/or secondary hyperosteogeny. The osteoarthritis refers to a chronic joint disease with joint cartilage degeneration and/or secondary hyperosteogeny as main pathological changes.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a combination of caladium and yam extract III and a new application thereof in the aspect of treating and preventing osteoarthritis. The two can not well inhibit the degradation of cartilage collagen when used alone, but can better inhibit MMP13 expression and promote COL2A1 expression when the two form a composition according to the mass ratio of 1:2, thereby inhibiting the IL-1 beta-induced degradation of cartilage matrix and showing stronger inhibition and protection effects on the degradation of the cartilage matrix of a mouse DMM model. Therefore, the combination of the caladium and the yam essence III can be used for medicines and health-care products which have main functions of preventing and treating osteoarthritis.

(IV) description of the drawings

FIG. 1 shows inhibition of Coelogyne, Yamamotoxin III and combinations thereof on ATDC5 cell growth and IL-1 beta-induced collagen degradation in ATDC5 cells; a is the inhibition effect of the fritillaria lancina on the growth of ATDC5 cells,^*indicates a significant difference from the control group, P<0.05; b is the growth inhibition effect of the dioscin III on ATDC5 cells; c is calanoline, yam essence III and their combination which can inhibit the growth of ATDC5 cell;^*indicates a significant difference from the control group, P<0.05，^**Indicates a very significant difference, P, from the control group<0.01；^##Representing a very significant difference, P, compared to the model group (IL-1. beta. treated alone)<0.01. D is the result of the drug treatment of ATDC5 cells, and the staining and photographing of the alisin blue after the IL-1 beta induction treatment; e, ATDC5 cell drug treatment, IL-1 beta induction, measurement of absorbance value at 620nm of guanidine hydrochloride leaching liquor after staining of Alisin blue, and calculation and analysis of collagen matrix degradation conditions;^*indicates a significant difference from the control group, P<0.05，^**Indicates a very significant difference, P, from the control group<0.01；^#Representing significant differences compared to the model group (IL-1. beta. alone treatment), P<0.05，^##Representing a very significant difference, P, compared to the model group (IL-1. beta. treated alone)<0.01。

FIG. 2, effect of composition on COL2A1(A) and MMP13(B) gene expression; note:^*the representation has significant differences, P, compared with the model group<0.05；^**The representation has significant differences, P, compared with the model group<0.01; # indicates significant difference compared to model group, P<0.01。

FIG. 3 effect of composition on COL2A1 and MMP13 gene expression; a is a Western blot development picture; b is the relative expression quantity of COL2A 1; c is relative expression quantity of MMP 13; note:^*the representation has significant differences, P, compared with the model group<0.05；^**The representation has significant differences, P, compared with the model group<0.01；^#Indicates a significant difference from the control group, P<0.05；^##Indicates a significant difference from the control group, P<0.01。

FIG. 4, results of safranin-fast green staining of sections of example 5.

FIG. 5, example 6 results of safranin-fast green staining of joint sections; note: A-D are sham surgery, model, composition low (40mg/kg) and composition high (80mg/kg) groups, respectively.

FIG. 6, example 6 relative expression levels of MMP13 and COL2A1 in articular cartilage; a is a Western blot development picture; b is relative expression quantity of MMP 13; c is the relative expression quantity of COL2A 1; note: denotes significant difference compared to model group, P <0.05, denotes very significant difference compared to model group, P < 0.01; # indicates a very significant difference compared to sham, P < 0.01.

(V) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

mouse chondroblasts (ADTC5), Shanghai Zeye Biotech, Inc., used in the examples of the present invention; male ICR mice, 21-25g in weight, Shanghai BK.

Drugs and reagents: the peiminine, the yam rhizome III and the BCA protein quantitative kit are all purchased from Shanghai source leaf biotechnology limited; BD-CBA mouse inflammatory factor detection kit (BD corporation, USA); DMEM medium, DMEM/F12 differentiation medium, fetal bovine serum were purchased from Gibco; ITS inducer (Sigma company); 1% aliskiren stain (solibao biotechnology limited); IL-1. beta. (Sino Biological Co.); reverse transcription kit (Takara corporation); protease and phosphatase inhibitors (Roche); M-PER mammalian protein extraction reagent (Thermo Fisher Scientific Co.); a full-automatic WB detection kit (12-230kDa separation 8X 25Capillary Cartridges, Protein simple Co.); Anti-Collagen II antibody (ab34712), Anti-MMP13 antibody (ab219620), Anti-GAPDH antibody (ab8245) antibody (Abcam Corp.); the primer is synthesized from Shanghai; chromatographically pure acetonitrile and formic acid (merck); other reagents were analytically pure.

The instrument comprises the following steps: fluorescent quantitative PCR instrument, CO₂Biological incubator, biological tissue paraffin embedding machine (seimer feishell science and technology); a multi-functional microplate reader (Perkin Elmer Co.); automated wes analytical systems (Protein simple, Inc.); rotary microtomes (Lecia Corp.); upright microscope (Motic Corp.).

In the embodiment of the invention, the calanine is prepared into mother liquor with the concentration of 25mg/mL by using dimethyl sulfoxide (DMSO), 40 mu L of the mother liquor is absorbed and added into 960 mu L of DMEM/F12 differentiation culture medium to be diluted into working solution with the concentration of 1mg/mL, and the working solution is added in the form of the working solution. The yamamizin III is prepared into mother liquor with the concentration of 25mg/mL by DMSO, 40 mu L of mother liquor is sucked and added into 960 mu L of DMEM/F12 differentiation medium to be diluted into working solution with the concentration of 1mg/mL, and the working solution is added in the form of the working solution.

Example 1 Effect of drugs on the growth of ADTC5 cells

(1) Grouping medicines: group 1: beimulanning; group 2: and (5) yam extract III.

(2) And (3) cell culture: taking ADTC5 cells in exponential growth phase, inoculating the cells into DMEM medium, and inoculating the cells in CO₂Biological incubator at 37 deg.C and CO₂Culturing under 5% condition until the cells are completely paved on the bottom of the bottle, changing into DMEM/F12 differentiation medium containing ITS inducer with mass concentration of 1%, continuously culturing, changing culture solution every 3d, and inducing differentiation culture for 21 d. After trypsinization, the cell density was adjusted to 5X 10 using DMEM/F12 differentiation medium⁵Each/mL was inoculated in 96-well plates, 200. mu.L per well.

(3) And (3) drug treatment: step (2) 96-well plate at 37 deg.C and 5% CO₂After overnight incubation under conditions, wells of the 96-well plate were divided into 2 groups. Group 1 was inoculated with varying concentrations of calanine (added as working solution at final concentrations of 0, 10, 20, 30, 40, 50 μ g/mL, 0 means addition of equal amounts of DMSO as reagent control), 3 wells per concentration; group 2 was inoculated with different concentrations of yamazarin iii (added as working solution at final concentrations of 0, 20, 30, 40, 50, 60 μ g/mL, 0 means that the same amount of DMSO was added as a reagent control group), 3 wells per concentration; adding the medicated 96-well plate at 37 deg.C and 5% CO₂After culturing for 48h under the condition, the cell survival rate is determined by a CCK-8 method.

(4) As a result: the results are shown in A and B in figure 1, and it can be seen from A in the figure that peiminine has no significant toxic effect on ADTC5 cells in the concentration range of 20-40 mug/mL, but the cell survival rate is reduced at the treatment concentration of 50 mug/mL, and the difference is significant compared with the reagent control treatment group; in FIG. 1, B shows that no significant inhibition of ADTC5 growth is observed in the concentration range of 20-60. mu.g/mL of dioscin III.

Example 2 inhibition of IL-1 β -induced degradation of collagen matrix in ADTC5 cells by drugs

(1) Grouping medicines: group 1: blank control, each group of drugs was replaced with an equal amount of DMSO; group 2: model group, each group of drug + IL-1 beta 10ng/mL was replaced with an equal amount of DMSO; group 3: different concentrations of calamine (10, 20, 40 mug/mL) + IL-1 beta 10 ng/mL; group 4: yam extract III (10, 20, 40 mug/mL) with different concentrations and IL-1 beta 10 ng/mL; group 5: the composition (Beimulaning 15 mu g/mL + Chinese yam rhizome extract III 15 mu g/mL, Beimulaning 10 mu g/mL + Chinese yam rhizome extract III 20 mu g/mL, Beimulaning 30 mu g/mL + Chinese yam rhizome extract III 10 mu g/mL, Beimulaning 20 mu g/mL + Chinese yam rhizome extract III 10 mu g/mL) and IL-1 beta 10 ng/mL.

(2) Cell culture: ADTC5 cells obtained after 21d of induced differentiation culture in the step (2) of example 1 were collected, and the cell density was adjusted to 5X 10 with DMEM/F12 differentiation medium⁵Each/mL was inoculated in 96-well plates, 200. mu.L per well.

(3) And (3) drug treatment: step (2) 96-well plate at 37 deg.C and 5% CO₂After overnight culture under the condition, grouping the medicines according to the step (1),adding medicine (fritillaria lanning and/or yam essence III) into each group, inoculating 200 μ L DMEM/F12 differentiation culture medium containing corresponding DMSO into each group 1 and 2, and culturing in 3-well; group 3 inoculated with varying concentrations of calanine, 3 wells per concentration; group 4 inoculated with different concentrations of dioscin III, each concentration being 3 wells; group 5 was inoculated with varying concentrations of calanoline + yam essence iii, 3 wells per concentration. Adding the medicated 96-well plate at 37 deg.C and 5% CO₂After pre-incubation for 2h under the conditions, groups 2 to 5 were divided into groups, and IL-1. beta. was added to each well at a final concentration of 10ng/mL, and CO was added at 37 ℃ to₂And (5) stimulating and culturing for 3 d. Removing supernatant, fixing with 95% methanol for 30min, rinsing with PBS for 3 times, adding 1% Alisin blue coloring agent, dyeing at room temperature for 30min, rinsing with PBS for 3 times, photographing, observing color depth, analyzing collagen degradation condition, and decreasing collagen degradation with darker color; then adding 200 mu L guanidine hydrochloride (6M) into each hole, soaking overnight, measuring the absorbance value at 620nm after oscillating and mixing uniformly, and comparing the absorbance values to analyze the degradation condition of the collagen matrix, wherein the higher the absorbance value is, the higher the collagen content is, and otherwise, the lower the collagen content is.

(4) As a result: the results are shown in FIG. 1C, which indicates that neither coerulenin nor yamazinol III can effectively inhibit IL-1 beta-induced collagen degradation in the range of 10-40 μ g/mL, respectively, in FIG. 1C; however, when the combination of the calanoline and the dioscin III is 15 mug/mL +15 mug/mL, 10 mug/mL +20 mug/mL and 7.5 mug/mL +22.5 mug/mL, the matrix degradation can be obviously inhibited compared with a model group (group 2: IL-1 beta single induction treatment group), the combination of 10 mug/mL +20 mug/mL is especially obvious and is almost equivalent to a normal control group (group 1); but when the proportion of the yam extract III or the calanine is continuously increased, the inhibition rate is reduced; therefore, the weight ratio of the calanoling to the yam essence III is 1:2, which is a better combination mode and has good inhibition effect on the collagen degradation of the chondrocytes induced by IL-1 beta.

Example 3 inhibition of IL-1 beta-induced degradation of collagen matrix in ADTC5 cells by drug concentration

(1) Grouping medicines: group 1: control, DMSO; group 2: model group, DMSO + IL-1 beta 10 ng/mL; group 3: composition 10. mu.g/mL + IL-1. beta.10 ng/mL; group 4: composition 20. mu.g/mL + IL-1. beta.10 ng/mL; group 5: composition 30. mu.g/mL + IL-1. beta.10 ng/mL; wherein the composition refers to the total amount of the caladium and the yam essence III mixed according to the mass ratio of 1: 2.

(2) Cell culture: ADTC5 cells were collected after 21d of the differentiation-inducing culture in the step (2) of example 1, and the cell density was adjusted to 5X 10 using DMEM/F12 differentiation medium⁵Each/mL was inoculated in 96-well plates, 200. mu.L per well.

(3) And (3) drug treatment:

step (2)96 pore plate at 37 deg.C and CO₂After culturing overnight under 5% condition, grouping according to the step (1) drugs, adding 200 μ L of DMSO containing corresponding concentration into groups 1 and 2; group 3 add composition 10 μ g/mL; group 4 add composition 20. mu.g/mL; group 5 was supplemented with 30. mu.g/mL of the composition. Adding the medicated 96-well plate at 37 deg.C and CO₂After 2 hours of preculture at 5% conditions, IL-1. beta. was added to each well of groups 2 to 5 in a final concentration of 10ng/mL at 37 ℃ in CO₂And (5) stimulating and culturing for 3 d. Removing supernatant, fixing with 95% methanol for 30min, rinsing with PBS for 3 times, adding 1% Alisin blue coloring agent, dyeing at room temperature for 30min, rinsing with PBS for 3 times, photographing, observing color depth, analyzing collagen degradation condition, and decreasing collagen degradation with darker color. Then 200 mu L of guanidine hydrochloride (6M) is added into each hole to be soaked at room temperature overnight, the absorbance value is measured at the position of 620nm after oscillation and uniform mixing, the degradation condition of the collagen matrix is analyzed by comparing the absorbance value, and the larger the absorbance value is, the higher the collagen content is.

As shown in D and E in FIG. 1, the proportion of the composition in the range of 10-30 μ g/mL was found to inhibit the IL-1 β -induced degradation of the collagen matrix in a dose-dependent manner, and the concentration of 20 μ g/mL was significantly different from that of the model group (group 2), and the treatment concentration of 30 μ g/mL was substantially equivalent to that of the control group (group 1), further confirming that the composition has a stronger collagen degradation inhibition effect at a mass ratio of 1:2 and a total treatment concentration of 30 μ g/mL (fritillaria laning 10 μ g/mL + dioscoreanin III 20 μ g/mL).

Example 4 Effect of compositions on collagen type II alpha 1(COL2A1) and matrix Metalloproteinase 13(MMP13) Gene expression

(1) Grouping medicines: different concentrations of the composition (0, 10, 20, 30 mug/mL) + IL-1 beta 10ng/mL, the composition refers to the total amount of the calanine and the yam III mixed according to the mass ratio of 1: 2; a blank control was also set up, and the composition and IL-1. beta. were not added, but replaced with DMSO containing an equivalent amount.

(2) Cell culture: ADTC5 cells cultured in induced differentiation of 21d in example 1 were collected, and the cell density was adjusted to 5X 10 in DMEM/F12 differentiation medium⁵Inoculate 12-well plates per mL, CO at 37 ℃₂After 5% overnight incubation, the compositions were added in groups of varying concentrations at 37 deg.C in accordance with step (1) with CO₂Pretreating for 2h under 5%, adding IL-1 beta with final concentration of 10ng/mL, and treating with CO at 37 deg.C₂Stimulating and culturing for 3d under the condition of 5 percent; the supernatant was removed and washed 2 times with pre-cooled PBS at 4 ℃.

(3) COL2a1 and MMP13 gene expression: the cells washed in the step (2) are subjected to Trizol method to extract total RNA, the total RNA is subjected to reverse transcription to form cDNA, fluorescent quantitative PCR is used for determining the expression conditions of COL2A1 and MMP13 genes (the primer sequences are shown in the table 1), the reaction system is shown in the table 2, and the results are shown in the figure 2.

TABLE 1 primer sequences

TABLE 2 fluorescent quantitative PCR reaction System

FIG. 2 shows that IL-1 beta induction can significantly reduce COL2A1 gene expression, and promote MMP13 gene expression; composition pretreatment dose-dependently up-regulated COL2a1 gene mRNA levels, especially in the 30 μ g/mL treated group even above the normal control group (see a in fig. 2); the pretreatment of the composition can also inhibit MMP13 gene expression in a dose-dependent manner, and the treatment groups with 20 mu g/mL and 30 mu g/mL have significant differences compared with the model group (see B in figure 2).

(4) Western blot analysis

After washing 2 times with PBS in step (2), the cells were lysed on ice for 30min according to the instructions of M-PER mammalian protein extraction kit (containing protease and phosphatase inhibitors) (Thermo Fisher Scientific, Waltham, MA, USA) with gentle shaking at variable times. The cell lysate was transferred to a 1.5mL EP tube and placed on ice and blown up with a 200. mu.L tip until the liquid was no longer viscous. The supernatant was carefully aspirated after centrifugation at 12000 rpm for 10min at 4 ℃ and the protein concentration was determined according to the BCA kit (leaf organisms of Shanghai origin) (B, C in FIG. 3).

WB analysis was performed in an automated wes analysis system (Protein simple, Inc.) using a fully automated WB detection kit (12-230kDa separation 8X 25Capillary Cartridges, Protein simple, Inc.) according to the kit instructions (A in FIG. 3). Anti-Collagen II antibody (ab34712), Anti-MMP13 antibody (ab219620), and Anti-GAPDH antibody (ab8245) were purchased from Abcam, diluted 1:50, and used according to the automated wes assay system protocol.

The Western blot results (FIG. 3) are substantially similar to the fluorescent quantitative PCR results, further indicating that the composition can reduce cartilage collagen matrix degradation by inhibiting matrix metalloproteinase expression, and can promote collagen matrix synthesis by up-regulating collagen type II synthesis gene COL2A1 expression, thereby acting from multiple layers and reducing cartilage damage.

Example 5 inhibition of IL-1 beta-induced degradation of articular cartilage collagen in mice by compositions in vitro

Carrying out cervical dislocation and sacrifice on ICR mice, sterilizing with 70% ethanol, dissecting and taking femoral head cartilage; rinsing with PBS containing double antibody (penicillin 100U/mL, streptomycin 0.1mg/mL) for 3 times, rinsing with sterile PBS, transferring into 96-well plate, adding 200 μ L DMEM culture medium into each well, and culturing at 37 deg.C under CO₂Pre-culturing for 2d under the condition of 5 percent; changing the culture solution, adding DMEM culture medium containing different concentrations of composition (0, 10, 20, 30 μ g/mL, wherein the composition is mixture of calanine and rhizoma Dioscoreae III at a mass ratio of 1:2, and 0 is DMSO with the same amount), and culturing at 37 deg.C under CO₂Pre-culturing for 2h under 5% condition, adding IL-1 beta with final concentration of 10ng/mL into each concentration composition, and adding CO at 37 deg.C₂3d stimulation at 5% conditions while setting blank controls without composition and IL-1 β, 6 cartilages were treated each. Fixing the treated cartilage with 10% neutral formalin for 48h, decalcifying and softening with 10% EDTA, washing with running water, slicing by conventional paraffin embedding,the degradation of cartilage collagen was observed by safranin-fast green staining and the results are shown in FIG. 4.

FIG. 4 shows the safranin-fast green staining result of the slice, the cartilage matrix in the normal group is stained uniformly and deep red, the staining of the outer edge of the slice stimulated by 10ng/mL IL-1 beta is obviously lightened or even completely whitened, and the degradation of the collagen matrix is serious; the border staining of the composition is obviously and gradually deepened under the treatment concentration of 10-30 mu g/mL, which shows that the composition can inhibit the degradation of cartilage matrix in a dose-dependent manner, and the treatment group with high dose of 30 mu g/mL is particularly remarkable.

EXAMPLE 6 protective Effect of the composition on mouse medial meniscal tibial ligament disassociation osteoarthritis (DMM)

1. Administration of drugs

Male ICR mice weighing 23 ± 2g, 48 in total, were divided into 4 groups (sham operation group, model group, composition low dose (40mg/kg) group and composition high dose (80mg/kg) group), the composition was a mixed total amount of calanine and yam rhizome iii in a mass ratio of 1:2, 12 in each group; injecting 2% sodium pentobarbital (40mg/kg) into abdominal cavity for anesthesia, shaving hair of right hind limb, cutting 1cm mouth on knee joint skin, opening joint cavity along patellar ligament with scalpel, carefully cutting medial meniscus tibial ligament under stereomicroscope, sterilizing, and suturing wound; the same procedure was performed in the sham group to open the joint space, but without severing the medial meniscotibial ligament, and the suture was sterilized and then sutured. Penicillin was injected intraperitoneally daily for 3 days after the operation to prevent infection, and administration was started 2 weeks after the operation. The pseudo-operation group and the model group were intraperitoneally injected with normal saline (containing 0.5% tween 80), and the composition low-dose group and the composition high-dose group were intraperitoneally injected with 40mg/kg and 80mg/kg compositions (wherein the compositions are prepared by mixing calanoline and yam rhizome III at a mass ratio of 1:2, dissolving with normal saline containing 0.5% tween 80, and preparing into composition solution with concentration of 5 mg/mL), and continuously administered for 6 weeks.

2. Safranin-fast green staining

Killing the mouse after cervical dislocation after 1h of last administration, dissecting and taking the knee joint, partially stripping the articular cartilage, and soaking and fixing the articular cartilage with 10% neutral formalin for 72 h; decalcifying with 10% EDTA decalcifying liquid for 30 days; and (3) dehydrating: performing gradient dehydration with anhydrous ethanol, 95%, 85%, and 70% ethanol for 5min each step; washing with water for 1min, dyeing with 0.02% fast green for 5min, dyeing with 1% acetic acid for 15s, dyeing with 0.1% safranin for 5min, washing with water for 30s, and dehydrating with anhydrous ethanol, 95%, 85%, and 70% ethanol for 1 min; carrying out xylene transparent treatment for 2 times, each time for 5 min; and sealing the neutral gum into pieces, observing through microscopic examination, and taking a picture for recording.

As can be seen from the safranin-fast green staining result in FIG. 5, the articular cartilage of the sham operation group is stained dark red and has a complete structure; the cartilage of the model group is light red, which indicates that the matrix is seriously degraded; the staining of the low-dose group is obviously deepened compared with that of the model group, and the high-dose group is almost equivalent to that of the sham operation group, which shows that the composition has a remarkable protective effect on the degradation of the cartilage matrix of the DMM model.

3. Western blot analysis

The articular cartilage is partially peeled off, frozen by liquid nitrogen and then stored at-80 ℃ for western blot analysis. Grinding frozen cartilage tissue with liquid nitrogen, adding 150 μ L precooled M-PER mammalian protein extraction reagent (containing protease and phosphorylase inhibitor), and lysing on ice for 30min while gently shaking for mixing. The cell lysate was transferred to a 1.5mL EP tube and placed on ice and blown with a 200 μ L tip until the liquid was no longer viscous. Centrifugation was carried out at 12000 rpm for 10min at 4 ℃ and the supernatant carefully aspirated, and the protein concentration was determined according to the BCA kit (leaf organisms of Shanghai origin). WB analysis was carried out according to the procedure of example 4 (4).

The Western blot result is shown in figure 6, and the result shows that the MMP13 expression amount in the sham operation group is lower, the MMP13 in the model group is obviously increased, and the MMP13 expression can be obviously reduced by 40mg/kg of the composition (P is less than 0.05); the high dose of 80mg/kg had a more significant inhibitory effect at a level comparable to that of the sham group. In contrast, the level of COL2a1 in the model group was significantly reduced, the degradation of COL2a1 in the composition-treated group with a high dose was significantly inhibited, and the degradation of COL2a1 in the composition-treated group with a low dose of 40mg/kg was not significantly inhibited as compared with the model group; the results are totally consistent with the results of section staining, and the results show that the composition can really inhibit the degradation of cartilage matrix by inhibiting the expression of MMP13 and promoting the expression of COL2A1 when being treated at 80mg/kg, better play the role of joint injury protection and show good development and application prospects.

Claims

1. The fritillaria and thunbergii composition for treating osteoarthritis is characterized by being prepared by mixing fritillaria and thunberg yam essence III according to a mass ratio of 1:1-1: 3.

2. The fritillaria thunbergii composition as claimed in claim 1, wherein the weight ratio of the fritillaria thunbergii to the yam rhizome iii is 1: 2.

3. The use of the fritillaria thunbergii composition of claim 1 in the preparation of a medicament for the treatment of osteoarthritis.

4. The use of claim 4 wherein the agent is an agent that inhibits the expression of matrix metalloproteinase 13 and simultaneously promotes the expression of type II collagen alpha 1.

5. The use of claim 4, wherein the medicament is a medicament for the treatment of articular cartilage degradation and/or secondary hyperosteogeny.