CN114557985B - A pharmaceutical composition for treating osteoarthritis and its application - Google Patents
A pharmaceutical composition for treating osteoarthritis and its application Download PDFInfo
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Abstract
The invention discloses a peimine composition for treating osteoarthritis and application thereof, wherein the peimine composition is prepared by mixing peimine and dioscin III in a mass ratio of 1:1-1:3. The invention discloses a fritillary and a preparation method thereof, which can well inhibit MMP13 expression and promote COL2A1 expression, thereby inhibiting IL-1 beta-induced cartilage matrix degradation and also has stronger inhibition and protection effects on mouse DMM model cartilage matrix degradation. Therefore, the combination of the invention can be used for medicines and health products with the main functions of preventing and treating osteoarthritis.
Description
Field of the art
The invention relates to a peiminine composition for treating osteoarthritis and application thereof.
(II) background art
Osteoarthritis (OA) is a common chronic degenerative joint disease with pain, swelling, and movement disorders as the main symptoms. Its pathogenesis is complex and diverse, not yet understood, but it is basically recognized that inflammatory responses play an important role in the initial stages of the disease. At present, nonsteroidal anti-inflammatory drugs are common drugs for treating OA, but only play a role in relieving joint pain, but do not improve joint degeneration, and long-term use can influence liver and kidney functions and increase the risk of cardiovascular events. The disease is well developed in middle-aged and elderly people, the life quality of the disease is seriously affected, and simultaneously, great burden is brought to medical treatment, sanitation and national economy. Thus, there is an urgent need to develop more effective medicines for treating OA.
Studies have shown that enhanced chondrocyte metabolic activity, and imbalance in extracellular matrix synthesis and catabolism are one of the important factors responsible for the pathogenesis of OA. In this process, overexpression of matrix metalloproteinases, such as matrix metalloproteinase 13 (MMP 13), is a key factor that leads to degradation of extracellular matrix, while reduction of extracellular matrix synthesis, such as insufficient synthesis of type ii collagen α1 (COL 2 A1), is another important factor that aggravates OA occurrence and development. Therefore, MMP13 and COL2A1 are important target molecules for developing OA therapeutic drugs, and a plurality of traditional Chinese medicine single and compound medicines with good treatment and prevention effects on OA show good regulation effects on the two target molecules, such as salvia miltiorrhiza extracts, peach kernel gonkang pills, qi-tonifying, stasis-tonifying and kidney-tonifying formulas and the like, and have good development and application prospects. However, the traditional Chinese medicine and the compound have the problems of unclear medicinal components, lack of index components, unstable curative effect and the like, so that the traditional Chinese medicine and the compound are necessarily standardized by adopting modern medicine technical means.
The research shows that the single action of the monomer components of the traditional Chinese medicine, namely the peiminine and the dioscin III, has no obvious inhibition effect on the degradation of the stroma of the chondrocyte induced by IL-1 beta, but the combination of the monomer components can obviously inhibit the degradation of the stroma; and further, in-vitro cartilage experiments and an in-vivo osteoarthritis model of mice prove that the composition with the mass ratio of 1:2 can inhibit MMP13 and promote COL2A1 expression, and has stronger inhibition and protection effects on IL-1 beta-induced cartilage matrix degradation and medial meniscus shin ligament dissociation osteoarthritis (DMM) cartilage matrix degradation. The therapeutic effect and molecular mechanism of the related compounds, namely, the peiminine, the dioscin III and the composition thereof in the aspect of osteoarthritis are not reported in the literature, and the compounds have obvious innovation.
(III) summary of the invention
The invention aims to provide a peimine composition for treating osteoarthritis and application thereof, wherein the peimine composition can inhibit MMP13 expression and promote COL2A1 expression at the same time, so as to achieve the effect of preventing or treating osteoarthritis, and the peimine and diosmin III have clear component structures and clear dose relation, so that the peimine composition has important significance in preparing OA therapeutic drugs with reliable quality and definite curative effect, and has wide application prospect in the aspect of treating osteoarthritis.
The technical scheme adopted by the invention is as follows:
the invention provides a peimine composition for treating osteoarthritis, which is prepared by mixing peimine and dioscin III in a mass ratio of 1:1-1:3, preferably 1:2.
The structural formulas of the peiminine and the dioscin III are as follows:
the peiminine and the dioscin III can be prepared by the prior known method.
The invention also provides application of the peimine composition in preparing medicines for treating osteoarthritis, wherein the medicines are medicines for inhibiting the expression of matrix metalloproteinase 13 (MMP 13) and promoting the expression of type II collagen alpha 1 (COL 2A 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 degeneration of articular cartilage and/or secondary hyperosteogeny as main pathological changes.
Compared with the prior art, the invention has the beneficial effects that:
the invention discloses a combination of peiminine and dioscin III and a new application thereof in the aspect of treating and preventing osteoarthritis. The two are used independently, but can well inhibit MMP13 expression and promote COL2A1 expression when the two are formed into a composition according to a mass ratio of 1:2, so that the degradation of cartilage matrix induced by IL-1 beta is inhibited, and the degradation of cartilage matrix of a mouse DMM model is also inhibited and protected strongly. Therefore, the combination of the invention can be used for medicines and health products with the main functions of preventing and treating osteoarthritis.
(IV) description of the drawings
FIG. 1, calamine, dioscin III and combinations thereof have inhibitory effect on ATDC5 cell growth and on IL-1 beta-induced collagen degradation of ATDC5 cells; a is the inhibition of ATDC5 cell growth by the fritillary bulb, * indicating significant differences compared with the control group, P<0.05; b is the inhibiting effect of dioscin III on ATDC5 cell growth; c is the inhibition of ATDC5 cell growth by the combination of the peiminine and the dioscin III; * indicating significant differences compared with the control group, P<0.05， ** Indicating a very significant difference, P, compared to the control group<0.01； ## Representing a very significant difference, P, compared to the model group (IL-1β alone)<0.01.D is the result of staining and photographing of the alisxin blue after ATDC5 cell drug treatment and IL-1 beta induction treatment; e is ATDC5 cell drug treatment, IL-1 beta induction, measuring absorbance value at 620nm of guanidine hydrochloride leaching solution after alisxin blue staining, and calculating and analyzing collagen matrix degradation condition; * indicating significant differences compared with the control group, P<0.05， ** Indicating a very significant difference, P, compared to the control group<0.01； # Representation of significant differences, P, compared to the model group (IL-1β alone)<0.05， ## Representing a very significant difference, P, compared to the model group (IL-1β alone)<0.01。
FIG. 2, effect of composition on COL2A1 (A) and MMP13 (B) gene expression; and (3) injection: * the representation has significant differences compared with the model group, P<0.05； ** The representation has significant differences compared with the model group, P<0.01; # indicates significant differences, P, compared to the model group<0.01。
FIG. 3, effect of composition on COL2A1 and MMP13 gene expression; a is a Western blot development chart; b is the relative expression quantity of COL2A 1; c is MMP13 relative expression quantity; and (3) injection: * the representation has significant differences compared with the model group, P<0.05； ** The representation has significant differences compared with the model group, P<0.01； # Indicating significant differences compared with the control group, P<0.05； ## Indicating significant differences compared with the control group, P<0.01。
FIG. 4, example 5, safranin-fast green staining results for sections.
FIG. 5, example 6 safranin-fast green staining results of joint sections; and (3) injection: A-D are sham surgery, model, low dose (40 mg/kg) and high dose (80 mg/kg) respectively.
FIG. 6 shows the relative expression levels of MMP13 and COL2A1 in articular cartilage of example 6; a is a Western blot development chart; b is the relative expression quantity of MMP 13; c is the relative expression amount of COL2A 1; and (3) injection: * Indicating significant differences compared to the model group, P <0.05, indicating significant differences compared to the model group, P <0.01; # indicates a very significant difference in P <0.01 compared to the sham surgery group.
(fifth) detailed description of the invention
The invention will be further described with reference to the following specific examples, but the scope of the invention is not limited thereto:
mouse chondroblasts (ADTC 5) used in the examples of the present invention, shanghai ze Biotechnology Co., ltd; male ICR mice, weighing 21-25g, shanghai BK company.
Medicine and reagent: the calamine, dioscin iii and BCA protein quantification kit were all purchased from shanghai source leaf biotechnology limited; BD-CBA mouse inflammatory factor detection kit (BD company of America); DMEM medium, DMEM/F12 differentiation medium, and fetal bovine serum were all purchased from Gibco corporation; ITS inducer (Sigma); 1% alisxin blue stain (soribao biotechnology limited); IL-1β (Sino Biological Co.); reverse transcription kit (Takara Co.); protease and phosphatase inhibitors (Roche); M-PER mammalian protein extraction reagent (Thermo Fisher Scientific company); full-automatic WB detection kit (12-230kDa separation 8X 25Capillary Cartridges,Protein simple company); anti-Collagen II antibody (ab 34712), anti-MMP13 antibody (ab 219620), anti-GAPDH antibody (ab 8245) antibody (Abcam corporation); the primer is synthesized by Shanghai technology; chromatographic pure acetonitrile and formic acid (merck); the other reagents were all analytically pure.
Instrument: fluorescent quantitative PCR instrument, CO 2 Biological incubator, biological tissue paraffin embedding machine (zemoeimer femil technologies); multifunctional enzyme-labeled instrument (Perkin Elmer company); automated wes analysis System (Protein sample Co.); rotary microtomes (Lecia corporation); front-mounted microscope (Motic).
In the embodiment of the invention, the peimine is prepared into a mother solution with the concentration of 25mg/mL by using dimethyl sulfoxide (DMSO), 40 mu L of the mother solution is absorbed, 960 mu L of DMEM/F12 differentiation medium is added to dilute the mother solution into a working solution with the concentration of 1mg/mL, and the working solution is added. The yam extract III is prepared into mother liquor with the concentration of 25mg/mL by DMSO, 40 mu L of mother liquor is absorbed, 960 mu L of DMEM/F12 differentiation medium is added to dilute the mother liquor into working solution with the concentration of 1mg/mL, and the working solution is added.
EXAMPLE 1 Effect of drugs on ADTC5 cell growth
(1) Grouping of drugs: group 1: fritillary bulb medicine; group 2: dioscin III.
(2) Cell culture: inoculating ADTC5 cells in exponential growth phase into DMEM culture medium, and inoculating into CO 2 37 ℃ and CO in a biological incubator 2 Culturing under 5% condition until the cells completely fill the bottle bottom, 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 21d. After pancreatin digestion, the cell density was adjusted to 5X 10 with DMEM/F12 differentiation medium 5 A96-well plate was seeded at a volume of 200. Mu.L per well.
(3) And (3) drug treatment: step (2) 96 well plate at 37℃in 5% CO 2 After overnight incubation under conditions, the wells of the 96-well plates were divided into 2 groups. Group 1 was inoculated with various concentrations of calamine (added as working fluid at final concentrations of 0, 10, 20, 30, 40, 50 μg/mL,0 refers to the addition of an equal amount of DMSO as a reagent control group), 3 wells per concentration; group 2 was inoculated with different concentrations of dioscin iii (added as working fluid at final concentrations of 0, 20, 30, 40, 50, 60 μg/mL,0 means adding an equal amount of DMSO as reagent control), 3 wells per concentration; the dosed 96-well plate was subjected to 5% CO at 37 ℃ 2 After 48h of incubation, the cell viability was determined using CCK-8.
(4) Results: the results are shown in figures 1A and B, and from figure A, the fritillary eupatorium has no obvious toxic effect on ADTC5 cells in the concentration range of 20-40 mug/mL, but the cell survival rate is reduced in the treatment concentration of 50 mug/mL, and the fritillary eupatorium has obvious difference compared with the reagent control treatment group; in figure 1, B shows that the dioscin III has no obvious inhibition effect on the growth of ADTC5 in the concentration range of 20-60 mug/mL.
EXAMPLE 2 inhibition of IL-1 beta induced degradation of ADTC5 cell collagen matrix by drugs
(1) Grouping of drugs: group 1: blank control, replace each group of drug with an equal amount of DMSO; group 2: model groups, each group of drugs +IL-1β10ng/mL was replaced with an equivalent amount of DMSO; group 3: different concentrations of calamine (10, 20, 40. Mu.g/mL) +IL-1β10ng/mL; group 4: different concentrations of dioscin III (10, 20, 40. Mu.g/mL) +IL-1β10ng/mL; group 5: composition (15. Mu.g/mL of calamine + 15. Mu.g/mL of dioscin III, 10. Mu.g/mL of calamine + 20. Mu.g/mL of dioscin III, 30. Mu.g/mL of calamine + 10. Mu.g/mL of dioscin III, 20. Mu.g/mL of calamine + 10. Mu.g/mL of dioscin III) +IL-1. Beta.10 ng/mL).
(2) Cell culture: ADTC5 cells after 21d of induced differentiation culture were obtained in step (2) of example 1, and the cell density was adjusted to 5X 10 with DMEM/F12 differentiation medium 5 A96-well plate was seeded at a volume of 200. Mu.L per well.
(3) And (3) drug treatment: step (2) 96 well plate at 37℃in 5% CO 2 After overnight incubation, the drug groups were followed by step (1) and each group was first added with drug (peiminine and/or diosmin iii), groups 1 and 2 were inoculated with 200 μl of DMEM/F12 differentiation medium containing the corresponding DMSO, and each group was 3 wells; group 3 was inoculated with different concentrations of calamine, 3 wells per concentration; inoculating different concentrations of dioscin III in group 4, wherein each concentration is 3 holes; group 5 inoculationDifferent concentrations of calamine + dioscin iii, 3 wells per concentration. The dosed 96-well plate was subjected to 5% CO at 37 ℃ 2 After 2h of preculture under the conditions, IL-1β was added to each well of groups 2 to 5 at a final concentration of 10ng/mL at 37℃in CO 2 Stimulated culture at 5% for 3d. Removing the supernatant, fixing with 95% methanol for 30min, washing with PBS for 3 times, adding 1% alisxin blue stain, dyeing at room temperature for 30min, washing with PBS for 3 times, photographing to observe collagen degradation condition with darker color, and degrading less collagen; then 200 mu L guanidine hydrochloride (6M) is added into each hole to soak overnight, absorbance value is measured at 620nm after shaking and mixing, the degradation condition of the collagen matrix is analyzed by comparing the absorbance value, the higher the absorbance value is, the higher the collagen content is, and the lower the collagen content is.
(4) Results: the results are shown as C in FIG. 1, and C in FIG. 1 shows that the peiminine and the dioscin III respectively fail to effectively inhibit the IL-1 beta-induced collagen degradation in the range of 10-40 mug/mL; however, when the combination of the peiminine 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, compared with the model group (group 2: IL-1 beta single induction treatment group), the combination of 10 mug/mL+20 mug/mL is particularly remarkable and almost equivalent to the normal control group (group 1); however, when the proportion of the dioscin III or the peiminine is continuously increased, the inhibition rate is reduced; therefore, the mass ratio of the peiminine to the dioscin 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 ADTC5 cell collagen matrix by drug concentration
(1) Grouping of drugs: group 1: control group, DMSO; group 2: model group, DMSO+IL-1β10ng/mL; group 3: 10 mug/mL of the composition plus 10ng/mL of IL-1 beta; group 4: 20 mug/mL of the composition plus 10ng/mL of IL-1 beta; group 5: 30 mug/mL of the composition plus 10ng/mL of IL-1 beta; wherein the composition refers to the total amount of the mixed peiminine and the yam extract III in a mass ratio of 1:2.
(2) Cell culture: ADTC5 cells after 21d of induced differentiation culture were obtained in step (2) of example 1, and the cell density was adjusted to 5X 10 with DMEM/F12 differentiation medium 5 one/mL of the cells were inoculated into 96-well plates,200. Mu.L of seed was inoculated per well.
(3) And (3) drug treatment:
step (2) 96 well plate at 37℃and CO 2 After overnight incubation at 5%, groups 1, 2 were supplemented with 200 μl of DMSO at the corresponding concentration according to step (1) drug group; group 3 added composition 10 μg/mL; group 4 add 20 μg/mL of composition; group 5 the composition was added at 30 μg/mL. The added 96-well plate is processed at 37 ℃ and CO 2 After preculture at 5% for 2h, IL-1β was added at a final concentration of 10ng/mL per well of groups 2 to 5 in groups, CO at 37℃ 2 Stimulated culture at 5% for 3d. Removing the supernatant, fixing with 95% methanol for 30min, washing with PBS for 3 times, adding 1% alisxin blue stain, dyeing with PBS for 30min at room temperature, washing with PBS for 3 times, photographing, observing the degradation condition of collagen by deep and light analysis, and the degradation of collagen is less when the color is deeper. Then 200 mu L guanidine hydrochloride (6M) is added into each hole to soak overnight at room temperature, absorbance value is measured at 620nm after shaking and mixing, the degradation condition of collagen matrix is analyzed by comparing the absorbance value, and the collagen content is higher as the absorbance value is larger.
As shown in the results of D and E in FIG. 1, the composition has obvious difference in the ratio of 10-30 mug/mL and the concentration of 20 mug/mL compared with the model group (group 2), and the treatment concentration of 30 mug/mL is basically equivalent to that of the control group (group 1), so that the composition has stronger collagen degradation inhibition effect at the mass ratio of 1:2 and the total treatment concentration of 30 mug/mL (10 mug/mL of peimine and 20 mug/mL of diosmin III).
Example 4 influence of compositions on type II collagen alpha 1 (COL 2A 1) and matrix metalloproteinase 13 (MMP 13) Gene expression
(1) Grouping of drugs: different concentrations of the composition (0, 10, 20, 30 mug/mL) +IL-1β10ng/mL, wherein the composition refers to the mixed total amount of the peimine and the dioscin III in a mass ratio of 1:2; a blank control group was also set up, without addition of composition and IL-1β, and replaced with DMSO containing an equivalent amount.
(2) Cell culture: ADTC5 cells of example 1 induced differentiation culture 21d were used to adjust cell density to 5X 10 with DMEM/F12 differentiation medium 5 Inoculating 12-well plate at 37deg.C、CO 2 After overnight incubation at 5%, the compositions of different concentrations were added in groups according to step (1) at 37℃with CO 2 Pretreating at 5% for 2 hr, adding IL-1β with final concentration of 10ng/mL, and CO at 37deg.C 2 Stimulated culture at 5% for 3d; removing the supernatant, and rinsing with PBS (phosphate buffered saline) at 4 ℃ for 2 times.
(3) COL2A1 and MMP13 Gene expression profiles: and (3) extracting total RNA from the washed cells in the step (2) by adopting a Trizol method, carrying out reverse transcription to obtain cDNA, and determining the expression conditions of COL2A1 and MMP13 genes by adopting fluorescent quantitative PCR (primer sequences are shown in Table 1), wherein a reaction system is shown in Table 2, and the result is shown in FIG. 2.
TABLE 1 primer sequences
TABLE 2 fluorescent quantitative PCR reaction System
The fluorescent quantitative PCR result of FIG. 2 shows that IL-1 beta induction can significantly reduce COL2A1 gene expression and promote MMP13 gene expression; composition pretreatment can dose-dependently up-regulate COL2A1 gene mRNA levels, especially in 30 μg/mL treated groups even above normal control groups (see fig. 2 a); composition pretreatment also inhibited MMP13 gene expression dose-dependently, with significant differences between the 20 μg/mL and 30 μg/mL treated groups compared to the model group (see B in FIG. 2).
(4) Western blot analysis
The cells after 2 times of PBS washing in the step (2) are processed according to the instructions of an M-PER mammalian protein extraction kit (containing protease and phosphatase inhibitor) (Thermo Fisher Scientific, waltham, MA, USA), and are lysed on an ice bath for 30min, and are mixed by gentle shaking at random. The above cell lysates were transferred to 1.5mL EP tubes, placed on ice and blown with a 200. Mu.L gun until the liquid was no longer viscous. Centrifugation at 12 rpm at 4℃for 10min, careful aspiration of the supernatant and determination of protein concentration (B, C in FIG. 3) was performed according to BCA kit (Shanghai derived leaf organism) instructions.
A full-automatic WB assay kit (12-230kDa separation 8X 25Capillary Cartridges,Protein simple) was used to perform WB assay (FIG. 3A) in an automated wes analytical system (Protein sample Co.) according to the kit instructions. Anti-Collagen II antibody (ab 34712), anti-MMP13 antibody (ab 219620), anti-GAPDH antibody (ab 8245) antibodies were all purchased from Abcam, diluted 1:50 and used according to the instructions of the automated wes analysis system.
Western blot results (FIG. 3) are substantially similar to fluorescent quantitative PCR results, further demonstrating that the composition can reduce cartilage collagen matrix degradation by inhibiting matrix metalloproteinase expression, and can promote collagen matrix synthesis by up-regulating type II collagen synthesis gene COL2A1 expression, acting from multiple levels, and reducing cartilage damage.
Example 5 inhibition of in vitro IL-1. Beta. Induced collagen degradation in mouse articular cartilage by compositions
ICR mice are killed by cervical dislocation, sterilized by 70% ethanol, and dissected to obtain femoral head cartilage; washing with PBS containing double antibody (penicillin 100U/mL, streptomycin 0.1 mg/mL) for 3 times, washing with sterile PBS, transferring into 96-well plate, adding 200 μl of DMEM medium into each well, and adding CO at 37deg.C 2 Pre-culturing for 2d under the condition of 5%; adding different concentration of composition (0, 10, 20, 30 μg/mL) after changing culture solution, wherein the composition is mixed by peiminine and dioscin III in a mass ratio of 1:2, 0 means adding equivalent DMSO), and adding CO at 37deg.C 2 Pre-culturing at 5% for 2 hr, adding IL-1β with final concentration of 10ng/mL into each concentration composition, and CO at 37deg.C 2 Stimulation was performed for 3d at 5% while a blank control without addition of composition and IL-1β was set, each for 6 cartilage treatments. The treated cartilage was decalcified and softened with 10% neutral formalin, 10% EDTA, washed with running water, sectioned by conventional paraffin embedding, and stained with safranin-fast green to observe collagen degradation in cartilage, and the results are shown in FIG. 4.
The safranin-fast green staining result of the section in FIG. 4 shows that the normal group cartilage matrix is uniformly stained in dark red, and the outer edge of the section is obviously shallowed or even completely whitened by the stimulation of 10ng/mL IL-1 beta, which indicates that the collagen matrix is seriously degraded; the edge 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 obvious.
EXAMPLE 6 protection of the composition against the medial meniscal shin ligament dissociative osteoarthritis (DMM) in mice
1. Administration of drugs
Male ICR mice weighing 23+ -2 g, 48 animals, and dividing into 4 groups (artificial operation group, model group, composition low dose (40 mg/kg) group and composition high dose (80 mg/kg) group), wherein the composition is mixed total amount of peimine and dioscin III in a mass ratio of 1:2, and each group comprises 12 animals; injecting 2% pentobarbital sodium (40 mg/kg) into abdominal cavity for anesthesia, shaving off the hair of right hind limb, cutting the skin at knee joint for about 1cm, opening joint cavity along patellar ligament with surgical knife, carefully cutting off inner meniscus shin ligament under stereomicroscope, sterilizing, and suturing wound; the sham operation group opens the joint cavity in the same operation, but does not break the medial meniscus shin ligament, and is sutured after disinfection. The penicillin is injected intraperitoneally every day within 3 days after the operation to prevent infection, and administration is started 2 weeks after the operation. The sham operation group and the model group are respectively injected with physiological saline (containing 0.5% Tween 80) and the low dose group and the high dose group of the composition are respectively injected with 40mg/kg and 80mg/kg of the composition intraperitoneally (wherein the composition is a mixture of the peiminine and the dioscin III in a mass ratio of 1:2, and the mixture is dissolved by the physiological saline containing 0.5% Tween 80 to prepare a composition solution with a concentration of 5 mg/mL) and the mixture is continuously administrated for 6 weeks.
2. Safranin-fast green dyeing
Killing the mice after cervical dislocation after the last administration for 1h, dissecting knee joints, partially stripping off articular cartilage, and soaking and fixing the mice with 10% neutral formalin for 72h; decalcification for 30 days with 10% EDTA decalcification solution; dehydrating: dehydrating with absolute ethanol, 95%,85% and 70% alcohol gradient for 5min; washing with water for 1min, dyeing with solid green of 0.02% for 5min, dyeing with acetic acid of 1% for 15s, dyeing with safranin of 0.1% for 5min, washing with water for 30s, and dehydrating with absolute ethanol of 95%,85% and 70% alcohol for 1min respectively; the xylene is treated for 2 times in a transparent way, and each time is 5min; and (5) sealing the neutral resin, performing microscopic examination, observing, and photographing and recording.
As can be seen from the safranin-fast green staining results of FIG. 5, the joint cartilage of the sham surgery group stained in dark red and has complete structure; the cartilage of the model group is light red, which indicates that the matrix is seriously degraded; the low dose of the administration group is obviously deepened compared with the model group, and the high dose group is almost equivalent to the false operation group, which shows that the composition has obvious protection effect on the degradation of the cartilage matrix of the DMM model.
3. Western blot analysis
And (5) after partial stripping of articular cartilage, quick freezing by liquid nitrogen, and storing at-80 ℃ for western blot analysis. Frozen cartilage tissue was taken, ground with liquid nitrogen, then 150 μl of pre-chilled M-PER mammalian protein extraction reagent (containing protease and phosphorylase inhibitor) was added, and the mixture was placed on ice for 30min of lysis, with gentle shaking and mixing at random. The above cell lysates were transferred to 1.5mL EP tubes, placed on ice and blown with a 200. Mu.L gun until the liquid was no longer viscous. Centrifugation at 12 rpm at 4℃for 10min, careful aspiration of the supernatant and determination of protein concentration was performed according to BCA kit (Shanghai derived leaf organism) instructions. WB analysis was performed according to the procedure of example 4 (4).
The Western blot results are shown in figure 6, and the results show that the expression level of MMP13 in a sham operation group is lower, the MMP13 in a model group is obviously increased, and 40mg/kg of the composition can obviously reduce the expression of MMP13 (P < 0.05); the high dose 80mg/kg inhibition was more pronounced at levels comparable to the sham operated group. In contrast, the COL2A1 level of the model group is obviously reduced, the COL2A1 degradation can be obviously inhibited by the high-dose treatment group of the composition, and the degradation can not be obviously inhibited by the low-dose 40mg/kg compared with the model group; the results are generally consistent with the slice staining results, and show that the composition can inhibit MMP13 expression and promote COL2A1 expression at the same time when being treated by 80mg/kg, so that cartilage matrix degradation is inhibited, joint injury protection is better exerted, and good development and application prospects are realized.
Claims (4)
1. A peimine composition for treating osteoarthritis is characterized by being prepared by mixing peimine and dioscin III in a mass ratio of 1:2.
2. Use of a combination of a peiminine according to claim 1 for the preparation of a medicament for the treatment of osteoarthritis.
3. The use according to claim 2, wherein the medicament is a medicament which inhibits matrix metalloproteinase 13 expression while promoting type ii collagen α1 expression.
4. The use according to claim 3, wherein the medicament is a medicament for the treatment of articular cartilage degradation and/or secondary hyperosteogeny.
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