CN110876753A - Application of pachyman in preparing medicine for preventing and treating rheumatoid arthritis - Google Patents

Abstract

The invention discloses an application of pachyman in preparing a medicament for preventing and treating rheumatoid arthritis, and also discloses a medicinal preparation for preventing and treating rheumatoid arthritis.A mouse collagen-induced arthritis model is applied to observe the treatment effect of pachyman on a mouse with rheumatoid arthritis, evaluate the change of proinflammatory cytokines IL-1 and TNF- α, observe the formation quantity of osteoclasts, and analyze various indexes of a CIA mouse, so as to clarify the treatment effect mechanism of the pachyman on the rheumatoid arthritis.cytokines in the rheumatoid arthritis, such as IL-1, TNF- α and the like can promote the activation of the osteoclasts to cause the erosion damage of bones and cartilages, are recognized as an important mechanism in the pathogenesis process of the rheumatoid arthritis at present, and the pachyman reduces the bone damage caused by the activation of the osteoclasts by inhibiting the expression of proinflammatory cytokines, so that the pachyman provides a new way for preventing and treating the rheumatoid arthritis.

Description

Application of pachyman in preparing medicine for preventing and treating rheumatoid arthritis

Technical Field

The invention belongs to the technical field of new application of chemical drugs, and particularly relates to application of pachyman in preparing drugs for preventing and treating rheumatoid arthritis.

Background

Rheumatoid Arthritis (RA) is a common chronic systemic autoimmune disease with chronic inflammation of joints as a major manifestation, and bone destruction is an important manifestation of RA. Overactivation of osteoclasts by increased in vivo proinflammatory cytokine secretion plays a key role in the progression of RA bone destruction. RA patients often cause articular cartilage and bone destruction due to persistent joint inflammation, finally cause joint deformity and loss of labor force, and bring serious burden to society and families. Statistically, about 7000 million people worldwide suffer from RA and a series of complications caused by RA, which bring serious economic burden to families and society. RA prevalence rate in continental areas of China is about 0.35-0.40%, but the prevalence rate is increased year by year.

The pathogenesis of RA is complex, the etiology is unknown, and factors such as heredity, inflammation and immunity play important roles. Traditional therapeutic drugs such as non-steroidal anti-inflammatory drugs and drugs for modifying the disease condition and resisting rheumatism have poor curative effect and poor prognosis. The rise of biological treatment (such as tumor necrosis factor antibody, interleukin receptor antibody and the like) and targeting small molecule preparation (such as JAK signal pathway inhibitor) brings hope for the treatment of RA, but has obvious curative effect on about 50 percent of patients. In recent years, the Chinese medicinal monomer is more and more widely applied to the treatment of the rheumatoid arthritis in China, has good curative effect both by oral administration and external treatment, and particularly has unique effects on relieving pain of patients and improving survival and treatment of the patients. Therefore, the monomer components of the traditional Chinese medicine with good curative effect and low side effect have important significance for treating the disease. The sinomenine, white peony root total combination and tripterygium glycosides which are used clinically at present are extracted from traditional Chinese medicines. The search for other effective traditional Chinese medicine monomers with good curative effect, small toxic and side effect and low cost has become a new hotspot for the research of the anti-rheumatoid arthritis medicine.

Pachymaran (Pachymaran), which is classified into water-soluble polysaccharides and alkali-soluble polysaccharides, is 50 β - (1 → 3) -bound glucose units, each β - (1 → 5) -bound glucose branch being separated from l to 2 β - (1 → 6) -bound glucose groups, and CAS No. SP 8930.

Pachyman (Pac) is the main component (about 90 percent of dry mass) in the dry sclerotium of the traditional Chinese medicine tuckahoe, the traditional Chinese medicine combines tuckahoe with other traditional Chinese medicines to treat diseases such as spleen deficiency, poor appetite, uneasy mind, loose stool, diarrhea, oliguria in water, palpitation, insomnia, phlegm and fluid retention and the like, wherein the tuckahoe represents cassia twig and tuckahoe soup, five-tuckahoe powder, four-monarch decoction and the like.

At present, no specific therapy exists for RA, research on pachymaran is insufficient, and reports on RA prevention and treatment by pachymaran monomers are not found, so that the invention is also provided.

Disclosure of Invention

The invention aims to provide a new application of pachyman (Pachymaran), in particular to an application of treating RA, and provides a new way for preventing and treating rheumatoid arthritis.

In order to achieve the purpose, the invention provides the following technical scheme:

application of pachyman in preparing medicine for preventing and treating rheumatoid arthritis is provided.

A pharmaceutical preparation for preventing and treating rheumatoid arthritis comprises pachyman or pachyman with effective amount for treatment and pharmaceutically acceptable adjuvants. The preparation method of the preparation adopts the conventional preparation method in the field to prepare the preparation on the premise of not reacting with the active ingredient of the invention or influencing the curative effect of the medicament.

Further, the pharmaceutical preparation is suitable for pharmaceutical preparations for gastrointestinal or parenteral administration. Furthermore, the pharmaceutical preparation is a freeze-dried preparation, an injection, a tablet, a granule or a capsule.

The dose of the pharmaceutical preparation of the present invention to be administered varies depending on the subject to be administered and the disease stage.

According to the technical scheme, whether Pac can have a treatment effect on a mouse arthritis (CIA) model induced by bovine type II collagen is researched by a gastric perfusion administration method, indexes such as Interleukin (IL) -1, IL-6, TNF- α and the like are measured at the same time, the relation between the effect and proinflammatory cytokines and bone destruction is researched by histological staining, and the mechanism and the theoretical basis of treating rheumatoid arthritis by pachyman are discussed.

According to the invention, 60 DBA/1 male mice in 6-8 weeks are randomly divided into a blank Control group (Control group), a CIA Model group (Model group), a low-dose pachyman treatment group (5 mg/kg/d) and a high-dose pachyman treatment group (10 mg/kg/d), and each group comprises 15 mice. After the CIA model was established according to the conventional method, 200. mu.L of physiological saline containing pachyman (wherein the doses of the low dose group and the high dose group were 5 mg/kg/d and 10 mg/kg/d, respectively) was administered daily by gavage from day 21 to the pachyman treatment group, and 200. mu.L of physiological saline was administered daily by gavage to the Control group and the model group. The animals of each group are sacrificed 42 days after the intragastric administration, the knee joints and the paw are taken, the Micro-CT and the histological examination are carried out, the bone erosion degree is evaluated, and the inflammatory bone destruction degree of the rheumatoid arthritis is analyzed by measuring the bone density, the bone volume fraction, the osteoclast expression, the expression of inflammatory factors and the like. Statistical analysis was performed on each group of data using one-way anova.

The invention has the beneficial effects that the therapeutic effect of pachyman on rheumatoid arthritis mice is observed by applying a mouse collagen-induced arthritis model, the change of proinflammatory cytokines IL-1 and TNF- α is evaluated, the formation quantity of osteoclasts is observed, and various indexes of CIA mice are analyzed, so that the therapeutic action mechanism of pachyman on rheumatoid arthritis is clarified.

Drawings

The invention is further described with reference to the following figures and examples.

FIG. 1 is a photograph of the paw of a mouse taken from each experimental group.

FIG. 2 is a graph showing the evaluation values of arthritis score and hind limb paw thickness in each experimental group of mice.

FIG. 3 is a three-dimensional model diagram of hind limb paw of each experimental group of mice scanned by micro-CT. A is Control group, B is Model group, C is Pac low dose treatment group, and D is Pac high dose treatment group.

FIG. 4 is a graph showing the measurement values of the hindfoot bone density (BMD) of mice in each experimental group.

FIG. 5 is a graph showing the detection values of the hindfoot bone volume fraction (BV/TV) of mice in each experimental group.

FIG. 6 is a graph showing the detection values of the hindfoot bone injury scores of the mice of each experimental group.

FIG. 7 is a graph showing the results of HE staining of knee joints of mice in each experimental group. A is Control group, B is Model group, C is Pac low dose treatment group, and D is Pac high dose treatment group.

FIG. 8 is a graph showing the detection values of the knee arthritis cell infiltration fraction of mice in each experimental group.

FIG. 9 is a chart showing the results of safranin O-fast green staining of knee joints of mice in each experimental group. A is Control group, B is Model group, C is Pac low dose treatment group, and D is Pac high dose treatment group.

FIG. 10 is a graph showing the detection values of the cartilage destruction scores of the mice in each experimental group.

FIG. 11 is a graph showing the results of staining the knee joint with tartrate-resistant acid phosphatase in mice of each experimental group. A is Control group, B is Model group, C is Pac low dose treatment group, and D is Pac high dose treatment group.

FIG. 12 is a graph showing the results of counting the cells stained positively by tartrate-resistant acid phosphatase in the knee joints of mice in each experimental group.

FIG. 13 is a graph showing the detection results of immunohistochemical staining of synovial IL-1 in knee joints of mice in each experimental group. A is Control group, B is Model group, C is Pac low dose treatment group, and D is Pac high dose treatment group.

FIG. 14 is a graph showing the positive quantitative results of the synovial IL-1 immunohistochemical staining of knee joints of mice in each experimental group.

FIG. 15 is a graph showing the results of immunohistochemical staining of synovial membrane TNF- α in knee joints of mice in each experimental group, wherein A is a Control group, B is a Model group, C is a Pac low-dose treatment group, and D is a Pac high-dose treatment group.

FIG. 16 is a graph showing the positive quantitative results of the immunohistochemical staining of synovial membrane TNF- α in knee joints of mice in each experimental group.

FIG. 17 is a graph of CTSK immunohistochemical staining detection results of knee joints of mice in each experimental group. A is Control group, B is Model group, C is Pac low dose treatment group, and D is Pac high dose treatment group.

FIG. 18 is a graph of the positive quantitative result of CTSK immunohistochemical staining of knee joints of mice in each experimental group.

FIG. 19 is a graph showing the result of MMP-9 immunohistochemical staining detection of the knee joint of mice in each experimental group. A is Control group, B is Model group, C is Pac low dose treatment group, and D is Pac high dose treatment group.

FIG. 20 is a graph showing the positive quantitative results of MMP-9 immunohistochemical staining of knee joints of mice in each experimental group.

Detailed Description

The present invention is further described below with reference to specific examples, which are only exemplary and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

Materials and methods

1. Material

1.1 reagent and Experimental Equipment

1.1.1 major drugs and reagents

Pachyman (Pachymaran, Pac), available from Solarbio, China, paraformaldehyde, sterile PBS, DAB color developer, hematoxylin, eosin, safranin, fast green, absolute ethanol, distilled water, 10% chloral hydrate, IL-1, TNF- α antibodies, available from Abcam, England.

1.1.2 Main Instrument

Micro-CT (SkyScan 1176, belgium), paraffin microtome (Leica 2135, germany), bake microtome (Leica 1120, germany), paraffin embedding machine (Leica 1150, germany), Axiovert 40C optical microscope (Zeiss, germany), surgical instrument suite, etc.

1.2 Experimental animals

60 healthy DBA/1 mice are male, have the weight of 20-25 g, are 6-8 weeks old and have the SPF grade, and are provided by the animal experiment center of Suzhou university. The breeding conditions were as follows: five cages are used, the room temperature is 18-20 ℃, the humidity is 50-60%, the ventilation is good, and the water can be freely taken.

2. Experimental methods

2.1 grouping of Experimental animals

60 DBA/1 mice were randomized into the following 4 groups:

(1) control group: 15, placebo group, from day 22 onwards, were sacrificed after gavage of 200 μ L saline daily and 42 consecutive days;

(2) model group: 15 mice, CIA model group, were fixed with a special holder with the tails exposed outside, and 0.1mL of emulsifier was injected intradermally at a distance of 1.5-2.0cm from the roots of the tails. The primary immunization injection adopts emulsifier mixed by complete Freund's adjuvant and collagen, and the primary immunization is strengthened by emulsifier mixed by incomplete Freund's adjuvant and collagen after 21 days. From day 22, 200 μ L of saline was gavaged daily and sacrificed after 42 consecutive days of injection;

(3) Low-Pac group: 10, a Pac low dose treatment group, in the same manner as model group modeling, were sacrificed after 42 consecutive days by gavage of 200 μ L of physiological saline containing 5mg/kg Pac from day 22 onward;

(4) High-Pac group: 10, the Pac high dose treatment group, in the same manner as the model group, were sacrificed after 42 consecutive days by gavage of 200. mu.L of physiological saline containing 10mg/kg Pac daily from day 22 onward.

2.2 preparation of mouse collagen-induced arthritis (CIA) model

The invention adopts a mouse CIA model induced by bovine type II collagen to simulate the pathological process of RA (Targetingpeptide inflammation and joint resolution in the mouse. J Clin invest.2002; 110(5): 611-2). The mouse is placed into a special mouse cage, the tail of the mouse can be exposed outside, an experimental assistant holds a special fixer to fix the root of the tail of the mouse, the left hand of the experimenter pinches the tail of the mouse, the right hand holds a Hamilton micro-injector to insert a needle at a position 1.5-2.0cm away from the root of the tail of the mouse, and 0.1mL of emulsifier is injected into the skin. During the bolus, spillage of the emulsifier should be avoided, and refilling should occur if spillage occurs. The first immunization injection adopts emulsifier mixed by complete Freund's adjuvant and collagen, and after 3 weeks, emulsifier mixed by incomplete Freund's adjuvant and collagen is used. The emulsifier doses for both injections were the same. The mice can have arthritis symptoms 4-5 days after the boosting immunization, the arthritis symptoms are particularly shown as red and swollen feet and paws with different degrees, the symptoms are the heaviest generally 35-38 days after the initial immunization, and the CIA modeling rate can reach about 95% 45 days after the initial immunization.

2.3 Scoring of mouse collagen-induced arthritis model

From 21 days after the initial immunization, the joint swelling degree of each group of mice was scored, and evaluated 2 times per week, 3 times per repetition of measurement, all measurements were independently performed by the experimenter and the experimental assistant, and the final results were averaged. Adopting an arthritis scoring method (0-4 points), wherein the point 0 is as follows: no arthritis; 1 minute; red spots or slight swelling; and 2, dividing: moderate swelling of the joint area; and 3, dividing: severe swelling; and 4, dividing: severe swelling and inability to bear weight, the sum of the limb scores is the arthritis index (maximum score 16).

2.4 specimen Collection

Mice were sacrificed 6 weeks after dosing, and bilateral hind limbs were retained, using the following specific procedures:

integrally separating knee joints and foot claws at two sides of a mouse, removing surface soft tissues, soaking the right hind limb in 10% paraformaldehyde solution for fixation for 48h, performing Micro-CT scanning, decalcifying with 10% EDTA, and performing histopathological detection; and (5) freezing and storing the left hind limb by liquid nitrogen.

2.5 Micro-CT detection

Scanning analysis of mouse hind limbs was performed using high resolution micro CT SkyScan1076 produced by SkyScan corporation, belgium. And (3) taking out the specimen from the fixing solution before scanning, and drying. Placing each sample in a Micro-CT test tube cup, wherein 3 samples are placed at a time, and each sample is separated by a foam plastic sheet; the specimens should be placed neatly to avoid touching the test tube wall. The scanning parameters are set as: the voltage is 70 kV, the current is 141 muA, the scanning time is 1750ms, the spatial resolution is 9μm, and after the scanning is finished, SkyScan1076 self-contained software is adopted to carry out three-dimensional reconstruction on the ankle joint of the mouse. The following parameters were analyzed using CT Analyzer analysis software (CT An, SkyScan): bone Density (BMD) in mg/cm³Represents; bone volume fraction (BV/TV;%); in mm^-1And (4) showing.

2.6 histological staining

After the decalcification of the knee joint specimen is finished by EDTA decalcification solution, trimming the specimen and reserving synovial tissue around the knee joint; finally, the paraffin-embedded specimen was sliced with a microtome to finally prepare a paraffin section having a thickness of 5 μm.

2.6.1 HE staining procedure:

(1) dewaxing the paraffin section by dimethylbenzene (10 min multiplied by 3 times), and then sequentially passing 100%, 95%, 90% and 85% ethanol to water, wherein each pass lasts for 5 min;

(2) washing with distilled water for 3min, staining with hematoxylin solution for 5min, and washing with tap water for 5 min;

(3) differentiating with 1% hydrochloric acid alcoholic solution for 60s, washing with tap water for 1 min;

(4) returning blue in 10% ammonia water solution for 60s, and washing with tap water for 1 min;

(5) counterstaining with l% eosin solution for 3min, and washing with tap water for 1 min;

(6) and (5) performing conventional dehydration, transparency and sealing.

Morphological changes of the synovial membrane and cartilage of the knee joint were observed under a light microscope. Synovial infiltration (Infiltratescope) and bone destruction (osteoporosis scope) were evaluated using a microscopy computer Image analysis system (Image-Proplus 6.0) with reference to the method of Hwang (Hwang JK, et al, Emodin rendering fluorescence responses and joint resolution in collagen-induced fracture analysis, Rheumatology (Oxford) 2013 Sep;52(9): 1583-91).

2.6.2 safranin O-fast Green staining:

(1) dewaxing the paraffin section by dimethylbenzene (10 min multiplied by 3 times), and then sequentially passing 100%, 95%, 90% and 85% ethanol to water, wherein each pass lasts for 5 min;

(2) washing with distilled water for 3min, staining with hematoxylin solution for 5min, and washing with tap water for 5 min;

(3) soaking in 0.02% fast green dye solution for 2 min;

(4) soaking the glass slide in 1% glacial acetic acid, and repeatedly removing the residual fast green dye on the glass slide for several times;

(5) dyeing for 2min by using l% safranin O dye liquor;

(6) and (5) conventionally dehydrating, transparentizing, sealing and collecting images under a microscope.

2.6.3 acid phosphatase resistant tartrate staining:

tartrate-resistant acid phosphatase (TRAP) is characteristic of osteoclasts and is distributed in the osteoclast cytoplasm. Under acidic conditions with tartrate, TRAP is able to hydrolyze naphthol ASBI phosphate to produce naphthol ASB1, which immediately binds to hexaazo-parafuchsin in the dye liquor to form an insoluble red dye at the enzyme active site. The acid phosphatase activity can be indirectly understood by observing this dye. TRAP staining was used to identify osteoclasts. Staining was performed using TRAP staining kit (Sigma 387A).

2.6.3.1 reagent preparation:

preparing 2 test tubes, adding 0.5mL fast Garnet GBC Base Solution (parafuchsin) in one test tube and 0.5mL LSodinium nitrile Solution (sodium Nitrite) in the other test tube, mixing for 30s, and standing for 2 min; preparing 2 100mL beakers, marking A and B, preparing TRAP dye solution (pH5.2):

reagent	A mL	B mL
			37 ℃ distilled water	45	45
Diazotized fuchsin	1.0	1.0
			Naphthol AS-BI Phosphate Solution	0.5	0.5
Acetate solution	2.0	2.0
			Tartrate solution	-	1.0

2.6.3.2 dyeing step:

(1) paraffin sections were deparaffinized and hydrated and washed 3 times for 3min with PBS

(2) Fixing the prepared specimen slices in an acetone solution for 30 s;

(3) washing with distilled water without drying;

(4) incubating TRAP dye liquor for 1 h at 37 ℃, and keeping out of the sun;

(5) distilled water was washed 3 times.

The positive TRAP staining results were purple red spots and lamellar areas, and the number of mature osteoclasts was counted under a 20 Xlight microscope field, with reference to the Nich C method (Nich C, et al, Roleof direct estrogen receptor signalling in near particulate-induced osteoclastogenesis. biomaterials.2013; 34(3): 641-50.).

2.6.4 immunohistochemical detection of CTSK, MMP-9, IL-1, TNF- α expression 1, Prior to deparaffinization and Hydrodeparaffinization, the sections were left to bake for 60 minutes at room temperature or 30 minutes at 60 ℃ in an incubator.

1) Soaking the slices in xylene for 10 minutes, and then soaking for 10 minutes after replacing the xylene; 2) Soaking in absolute ethyl alcohol for 5 minutes; 3) Soaking in 95% ethanol for 5 min; 4) soaking in 70% ethanol for 5 min; 2. antigen retrieval

The enzyme digestion method comprises the following steps: 0.1% trypsin is commonly used, the temperature of the trypsin is preheated to 37 ℃ before use, the slices are also preheated to 37 ℃, 0.2mL of digestive juice is dripped into each slice to cover the complete tissue, and the slices are digested in an incubator at 37 ℃ for about 5-30 minutes and are protected from light. 3. Immunohistochemical staining 1) washing with PBS for 2-3 times and 5 minutes respectively; 2) Normal goat serum confining liquid is added dropwise, and the temperature is 20 minutes. And throwing off the redundant liquid. 3) Mu.l of I-antibody was added dropwise and allowed to stand at room temperature for 1 hour, or overnight at 4 ℃ or 1 hour at 37 ℃. 4) After 4 ℃ overnight, the temperature was rewarmed at 37 ℃ for 45 minutes. 5) PBS wash 3 times for 5 minutes each; 6) Dripping 40-50 mu l of II antibody, and standing at room temperature or at 37 ℃ for 1 hour; 7) PBS wash 3 times for 5 minutes each; 8) DAB color development is carried out for 5-10 minutes, and the dyeing degree is grasped under a microscope; 9) Rinsing with PBS or tap water for 10 minutes; 10) Counterstaining with hematoxylin for 3 minutes, and differentiating with hydrochloric acid and alcohol; 11) Washing with tap water for 10-15 minutes; 12) Dehydrating, transparentizing, sealing and microscopic examination.

2.7 statistical analysis

The result data is analyzed by SPSS11.0 statistical software, and the data is averaged plus or minus standard deviation (

) It is shown that one-way ANOVA (one-way ANOVA test) is selected for the multiple groups of comparison, and LSD and Dunnett-t methods are selected for the two-by-two comparison under the condition of uniform overall variance.p<A difference of 0.05 is statistically significant.

Second, result in

1. Changes in the general conditions of the mice

1.1 general conditions and paw swelling of the experimental animals

In the whole experimental process, the mice in the blank Control group (Control group) have good mental state, can normally crawl and have no joint red and swollen; after secondary immunization, all mice of the CIA model group have typical arthritis symptoms, which are specifically manifested as red and swollen feet and paws, lusterless hair, disorder and sparseness, abnormal gait, poor mental state and irritability, swelling of feet with different degrees, thickened joints and red and swollen mucosa which are exuded; the diet condition of the mice in the Pac treatment group is obviously better than that of the mice in the model group, the mental state is better, the joints are slightly red and swollen, and no obvious movement disorder exists. During the molding period, no 1 death occurred in each group of mice. Wherein, A is a Control group, B is a CIA model group, C is a Low-Pac treatment group (5 mg/kg Pac), and D is a High-Pac treatment group (10 mg/kg Pac) as shown in figure 1.

1.2 mouse arthritis and hind foot thickness score

The results showed that the arthritis scores of the mice in the model group peaked at D42, with 6 of them having a score of 16, with an average score of 13.6 ± 1.53, and the difference was statistically significant (x) compared to the Control group (0)p<0.01); mice with no 16 arthritis score appeared in Pac treatment group D42, and the differences were statistically significant compared to the model group (.)p<0.05). See fig. 2. The hind limb paw thickness of the model group mice is obviously increased after the model secondary immunization, the average thickness in the peak period is 3.3 +/-0.3 mm, and compared with the Control group, the difference has statistical significancep<0.01); the differences between the Pac-treated group and the model group were statistically significant (.)p<0.05). See fig. 2.

Micro-CT detection

The Micro-CT scanning experiment mouse is used for foot paw and ankle joint and three-dimensional image reconstruction and quantitative analysis are carried out, so that the bone mass and bone microstructure can be accurately described, and the degree of arthritis damage is judged. Wherein, A is a Control group, B is a CIA model group, C is a Low-Pac treatment group (5 mg/kg Pac), and D is a High-Pac treatment group (10 mg/kg Pac). Displaying a three-dimensional reconstruction image, wherein the bone surface of a mouse in a Control group is smooth, the joint clearance is normal, and the joint structure is complete; compared with the Control group, the joint clearance of the mice in the CIA group is obviously narrowed or even disappears, the surface of the bone is rough, the obvious worm erosion-like change is generated, and the joint structure is seriously damaged; after administration of Pac, the joint space was significantly widened and bone surface destruction was significantly improved. See fig. 3.

Bone density (BMD): the bone density was significantly reduced in the model group compared to the Control groupp<0.01. After the Pac injection treatment, bone density gradually increased back with increasing Pac treatment amount, wherein the high dose Pac treatment group was compared to the model groupp<0.01, low dose Pac treatment group compared to model groupp<0.05. See fig. 4.

Bone volume fraction (BV/TV): the number of bone body integrals of the model group is obviously reduced, compared with the Control groupp<0.01. And the number of bone body scores increased significantly after Pac treatment, wherein high dose Pac treatment group was compared to model groupp<0.01, low dose Pac treatment group compared to model groupp<0.05. See fig. 5.

Bone injury scoring: the bone destruction was severe and the bone damage score was significantly increased in the model group, compared to the Control groupp<0.01. Decreased bone injury score in the treated groups following Pac treatment, wherein high dose Pac treated groups compared to model groupsp<0.01, low dose Pac treatment group compared to model groupp<0.05. See fig. 6.

3. Histological examination

3.1 HE staining results:

under a light microscope, the knee joint of the Control group mouse has complete joint surface, the joint gap exists, inflammatory cells do not infiltrate, and the phenomenon of synovium hyperplasia and thickening is not seen; the articular surface of the knee joint of the simulated mice is damaged, the joint space is narrowed or even disappears, the synovial membrane is seriously proliferated, and a large amount of inflammatory cells infiltrate; the synovial tissue of the Pac treatment group is slightly inflamed, a small amount of inflammatory cells infiltrate, pannus formation is not seen, and the joint space is slightly narrowed. Wherein, A is a Control group, B is a CIA model group, C is a Low-Pac treatment group (5 mg/kgPac), and D is a High-Pac treatment group (10 mg/kg Pac). See fig. 7.

Inflammatory cell Infiltration score (Infiltration score): compared with the Control group, the CIA model group (3.4 +/-0.5 mm) has obviously larger infiltration fraction and the difference has statistical significancep<0.01). After Pac treatment, infiltration scores were 2.5. + -. 0.4mm (group L) and 1.2. + -. 0.2mm (group H), respectively, and the differences were statistically significant compared to the CIA model groupp<0.05，**p<0.01). See fig. 8.

3.2 safranin O-fast Green staining

The knee joint surface of the mouse in the Control group is complete, the joint clearance is normal, and the safranin O is obviously colored; the joint clearance of the model group is obviously narrowed, and the safranin O staining is lost, which indicates that a large amount of cartilage is destroyed on the joint surface, and the synovial membrane hyperplasia and inflammatory cell infiltration are obvious; the Pac treatment group has joint gaps, more safranin O staining exists, articular surface cartilages are more complete, the thickness of local cartilaginous layer is thinner, synovial membranes are slightly hyperplastic, and a small amount of inflammatory cells infiltrate. Wherein, A is a Control group, B is a CIA model group, C is a Low-Pac treatment group (5 mg/kg Pac), and D is a High-Pac treatment group (10 mg/kg Pac) as shown in figure 9.

Cartilage destruction score: model group cartilage destruction score under light microscopy compared to Control groupp<0.01; while the cartilage destruction scores in the low and high concentration treatment groups of Pac were 1.5. + -. 0.3 and 0.9. + -. 0.3, respectively. The difference between the PAC high dose treatment group and the model group was statistically significant (. multidot.p<0.01), the differences between the Pac low dose treatment group and the model group were statistically significant (.)p<0.05). See fig. 10.

3.3 TRAP staining

The TRAP staining positive area is mauve, and the model group can see a large mauve area, which indicates that a large amount of mature osteoclasts exist in the bone tissue of the model group; control group only seen a very small number of purple-red positive areas; after administration (Low-Pac group, High-Pac group) the dark areas were significantly reduced, and only a few purple-red positive areas were visible. See fig. 11.

The under-light counting result showed that model group TRAP positive cells were 84. + -.11, compared with Control group (26. + -.5)p<0.01; and TRAP cell numbers of the Pac low-concentration treatment group and the Pac high-concentration treatment group are 60 +/-10 and 39 +/-9 respectively. The differences between the Pac high dose treatment group and the model group were statistically significant (. alpha.)p<0.01), the differences between the Pac low dose treatment group and the model group were statistically significant (.)p<0.05). See fig. 12.

4 immunohistochemical detection

The immunohistochemistry results showed that the expression level of IL-1 was observed under an optical microscope, as shown in FIG. 13. And Control group (17. + -. 4 mm)^-2) Comparison, model group (60. + -.7 mm)^-2) The expression of IL-1 is obviously increased, and the difference has statistical significancep<0.01). After Pac treatment, the IL-1 expression levels were 25. + -. 4mm, respectively^-2(Low-Pac group), 22. + -. 4mm^-2(High-Pac group), Pac High dose treatment group was statistically different from model group (x;)p<0.01), the differences between the Pac low dose treatment group and the model group were statistically significant (. + -)p<0.01). See fig. 14.

Further determination of the expression level of TNF- α was performed, as shown in FIG. 15, in combination with Control group (11. + -. 3 mm)^-2) Comparison, model group (48. + -.6 mm)^-2) The expression of TNF- α was significantly increased, and the difference was statistically significantp<0.01) TNF- α expression levels were 27. + -. 4mm after Pac treatment^-2(Low-Pac group), 19 +/-3 mm- (High-Pac group), and statistical difference between the Pac High-dose treatment group and the model groupp<0.01), the differences between the Pac low dose treatment group and the model group were statistically significant (. + -)p<0.01). See fig. 16.

CTSK staining results showed that, see FIG. 17, and Control group (16. + -.3 mm)^-2) Comparison, model group (48. + -.5 mm)^-2) The expression quantity is obviously increased and the difference isStatistical significance (. about.p<0.01). After Pac treatment, the CTSK expression quantity is 34 +/-5 mm^-2(Low-Pac group), 28. + -. 3mm^-2(High-Pac group), Pac High dose treatment group was statistically different from model group (x;)p<0.01), the differences between the Pac low dose treatment group and the model group were statistically significant (. + -)p<0.01). See fig. 18.

The result of MMP-9 staining is shown in FIG. 19, and is compared with Control group (18. + -. 3 mm)^-2) Comparison, model group (47. + -.5 mm)^-2) The expression quantity is obviously increased, and the difference has statistical significancep<0.01). After Pac treatment, the CTSK expression quantity is 39 +/-4 mm^-2(Low-Pac group), 27. + -. 4mm^-2(High-Pac group), Pac High dose treatment group was statistically different from model group (x;)p<0.01), the differences between the Pac low dose treatment group and the model group were statistically significant (.)p<0.05). See fig. 20.

The above test results are summarized as follows:

compared with the Control group, the mouse paw of the CIA model group gradually shows arthritis expressions such as erythema, swelling, deformity, limited movement and the like, the paw thickness and the arthritis score are obviously increased, and compared with the Control group, the difference has statistical significance (p is less than 0.01); after Pac treatment, the mice showed significantly reduced arthritic manifestations such as paw swelling, deformity, etc., with paw thickness and arthritic score decreasing significantly with increasing Pac concentration, p <0.01 compared to the CIA model group.

Micro-CT three-dimensional reconstruction results show that the paw bone density and the bone integration number of the model group are obviously reduced (p is less than 0.01) compared with the Control group; significant increase in bone injury score (. p < 0.01); the paw bone density and the bone body integral number in the Pac treatment group are obviously increased, the bone damage score is obviously reduced, and compared with the CIA model group, the star p is less than 0.01, and the effect is more obvious particularly in the high-dose treatment group.

The immunohistochemical results show that the positive expression of IL-1 and TNF- α is low in the Control group, the positive expression of IL-1 and TNF- α is sharply increased in the CIA model group, compared with the Control group, p is less than 0.01, the positive expression of IL-1 and TNF- α is obviously reduced along with the increase of the Pac concentration in the Pac treatment group, and meanwhile, the expression of the osteoclast activation related functional proteins CTSK and MMP-9 is also reduced along with the increase of the Pac concentration along with the reduction of the expression of proinflammatory cytokines.

The animal experiment proves that the pachymaran has better prevention and treatment effects on collagen-induced arthritis, can inhibit a large amount of proinflammatory cytokines secreted by monocytes, macrophages, synovial fibroblasts and the like, obviously inhibits the formation of osteoclasts, and can be used as a new means for medicine intervention on rheumatoid arthritis.

In summary, the following conclusions can be drawn through the above test results:

the experiment intervenes in a CIA mouse model by a mode of stomach-filling administration of pachymaran (Pac), and results show that the joint swelling degree of a treatment group is reduced, the arthritis score is reduced, the expression quantity of proinflammatory cytokines IL-1 and TNF- α in joint synovium is reduced, particularly, the effect of a high-dose group is more obvious, the expression of osteoclast-related CTSK and MMP-9 proteins can be inhibited, the number of osteoclasts is reduced, bone erosion is obviously improved, collagen-induced arthritis is inhibited, and the expression of IL-1 and TNF- α in bone tissues is reduced.

According to the above results, it is known that the action mechanism of Pac on rheumatoid arthritis is related to the inhibition of inflammatory factors such as IL-1, TNF- α and the formation of osteoclasts, IL-1 and TNF- α are mainly secreted by monocytes and macrophages, and can induce synovial cells to generate inflammatory factors such as matrix metalloproteinase and PGE2 in a large amount, so that extracellular matrix is damaged, and the cytokines can also induce the organism to generate an autoinflammatory reaction loop, so that the formation and activation of osteoclasts are promoted, and bone and articular bones and synovial tissues are continuously damaged.

Claims (4)

1. Application of pachyman in preparing medicine for preventing and treating rheumatoid arthritis is provided.

2. A pharmaceutical preparation for preventing and treating rheumatoid arthritis is characterized by comprising pachyman or pachyman with effective treatment amount and pharmaceutically acceptable auxiliary materials.

3. The pharmaceutical formulation of claim 2, wherein the pharmaceutical formulation is suitable for parenteral administration.

4. The pharmaceutical preparation of claim 2, wherein the pharmaceutical preparation is a lyophilized preparation, an injection, a tablet, a granule or a capsule.

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