Application of periploca forrestii schltr active cardiac glycoside monomer in preparation of medicine for treating rheumatoid arthritis

Abstract

The invention belongs to the field of medicines, and discloses application of periplocin and periplocin in preparation of medicines for treating rheumatoid arthritis. Periplocin can act on a bone morphogenetic protein-2 (BMP-2) signal channel through sodium / potassium-ATPase (NKA), and the expression quantity of osteogenesis-related protein is up-regulated, so that proliferation and differentiation of osteoblasts are promoted. The periplocin is orally taken, so that the foot swelling degree and joint injury of a rat RA model can be relieved in a dose-dependent manner, the erosion of a joint bone microstructure is relieved, the levels of various inflammatory factors in serum are inhibited, and the RA symptom is effectively improved. The invention provides a new direction for seeking safe and effective medicines capable of radically treating RA.

Description

Technical Field

[0001] The invention belongs to the field of medicine and relates to the application of active cardiac glycoside components of Achyranthes bidentata in preparing drugs for treating rheumatoid arthritis, in particular to the application of periplocoside and periplocoside aglycone in preparing drugs for treating rheumatoid arthritis. Background Art

[0002] Rheumatoid arthritis (RA) is a systemic autoimmune disease associated with a chronic inflammatory process. The number of patients accounts for about 1.0% of the world's total population. By 2025, the number of patients is expected to reach 317 million. [1] The onset of RA may be related to genetics, infection, sex hormones, etc. Its basic pathological characteristics are synovitis accompanied by destruction of articular cartilage and bone, which eventually leads to joint deformity and loss of function. It is also accompanied by many complications, such as various cardiovascular diseases, lung diseases, metabolic diseases, osteoporosis, Felty syndrome, etc. [2] In traditional Chinese medicine, rheumatoid arthritis is classified as "bi syndrome". [3] The disease has a long course and is stubborn. In mild cases, it affects joint function and in severe cases, it causes disability. It is called "immortal cancer."

[0003] Given the pathological characteristics of RA, the current best treatment strategy is to simultaneously treat joint inflammation and promote bone reconstruction. Bone reconstruction is a "renewal" process in which new bone replaces old bone. Osteoclasts (OCs) absorb old bone and osteoblasts (OBs) form new bone, thereby maintaining the bone balance of a healthy human body. Among them, promoting the differentiation and proliferation of OBs is an important means to regulate the bone reconstruction process and promote bone repair. [4,5] . Current treatments for RA are mainly anti-inflammatory, including methotrexate (MTX), glucocorticoids, and monoclonal antibodies targeting cytokines. Although these drugs can effectively delay the disease, their toxic effects, especially the side effects of long-term use, cannot be ignored. For example, methotrexate has a slow onset of action and significant side effects, which may lead to bone marrow suppression, liver damage, and liver fibrosis; long-term use of glucocorticoids may lead to increased blood sugar, osteoporosis, and liver damage; although biological inhibitors are effective, there are unpredictable adverse reactions and safety risks, such as headaches, skin ulcers, stomatitis, fever, and severe allergic reactions. [6] , and the high price brings a heavy economic burden to patients. In comparison, traditional Chinese medicine has the advantages of multiple targets, good efficacy, few side effects, and economical affordability. It has always been a research hotspot for the treatment of rheumatoid arthritis. Now, a variety of RA treatment drugs have been developed from traditional anti-rheumatic and anti-inflammatory Chinese medicines, such as Tripterygium wilfordii polyglycosides tablets, Sinomenine injection, White Peony Glucosides Capsules and Biqi Capsules [7] wait.

[0004] Periploca forrestii Schltr. is a plant of the genus Periploca in the Apocynaceae family. It grows in sunny places in sparse forests in mountains below 2,700 meters above sea level or under shaded and wet mixed woods or bushes. It is mainly distributed in Tibet, Qinghai, Sichuan, Guizhou, Guangxi and other places. Periploca forrestii was first recorded in the "Southern Yunnan Materia Medica". It is a common medicinal material used by the Miao people. The whole plant can be used as medicine and is known as one of the "three treasures" of Guizhou Miao medicine. The main chemical components of Periploca forrestii include cardiac glycosides, terpenes, flavonoids, phenylpropanoids and quinones. [8] It has multiple biological activities such as cardiotonic, anti-tumor, and immunosuppressive.

[0005] References:

[0006] [1] Black RJ, Brooks PM, Kopansky-Giles DR, et al. Global, regional, and national burden of rheumatoid arthritis, 1990-2020, and projections to 2050: systematic analysis of the Global Burden of Disease Study 2021[J]. The Lancet. Rheumatology, 2023, 5(10): 594-610.

[0007] [2]Wu D, Luo Y, Li T, et al. Systemic complications of rheumatoidarthritis: Focus on pathogenesis and treatment[J]. Front Immunol, 2022,13:1051082.

[0008] [3] Lan Peimin, Wang Pingren. Comparison of the etiology and pathogenesis of rheumatoid arthritis between traditional Chinese medicine and western medicine [J]. Chinese Folk Therapy, 2004, 12(12): 60-61.

[0009] [4]Boyle WJ,Simonet WS,Lacey DL.Osteoclast differentiation and activation[J].Nature,2003,423(6937):337-342.

[0010] [5]Billiard J, Moran RA, Whitley MZ, et al. Transcriptional profiling of human osteoblast differentiation[J]. Journal of Cellular Biochemistry, 2003, 89(2):389-400.

[0011] [6]Smolen JS,Aletaha D,Koeller M,et al.New therapies for treatment of rheumatoid arthritis[J].Lancet,2007,370(9602):1861-1874.

[0012] [7]Qu B, Wang XL, Zheng DC, et al. Treatment for refractory rheumatoidarthritis with total glucosides of paeony and nobiletin codelivered in a selfnanoemulsifying drug delivery system [J]. Acta Pharmacologica Sinica, 2022, 43: 2094-2108.

[0013] [8]Chen L, Tang S, Li Summary of the invention

[0014] The inventors used mouse embryonic cranial fibroblasts (MC3T3-E1) cells as a source of osteoblasts, and screened the in vitro osteoblast proliferation and differentiation activities of five representative cardiac glycoside monomers (periploca forrestii Schltr.) (periploca glycoside, periploca aglycone, periploca glycoside, digitoxin aglycone, and isoperiploca glycoside). The results showed that periploca glycoside and periploca aglycone had strong osteoblast proliferation and differentiation abilities, and the EC activity of periploca glycoside in promoting osteoblast proliferation was significantly higher than that of periploca glycoside. 50The value is 0.6nM, which promotes osteoblast differentiation 50 The value was 0.98nM, which was the lowest value among the five cardiac glycosides, preliminarily indicating that glycosides have good ability to promote proliferation and differentiation. The osteogenic proteins osteocalcin (OCN), osteoprotegerin (OPG), and type I collagen (COL-I) were used as detection indicators and combined with lentiviral transfection technology to further determine the osteogenic differentiation ability and related mechanisms of glycosides. It was found that glycosides can act on sodium / potassium-ATPase (Na + / K + -ATPase, NKA), activating the bone morphogenetic protein-2 (BMP-2) signaling pathway to promote osteodifferentiation. In the collagen-induced arthritis (CIA) rat RA model, oral administration of periploside can reduce the degree of foot swelling and joint damage in RA rats in a dose-dependent manner, promote bone reconstruction, and inhibit the levels of multiple inflammatory factors in serum.

[0015] The purpose of the present invention is to provide the use of periplocoside and periplocoside aglycone in the preparation of a drug for promoting osteocyte proliferation.

[0016] Another object of the present invention is to provide the use of periplocoside and periplocoside aglycone in the preparation of drugs for promoting osteocyte differentiation.

[0017] Preferably, the application is the application of periplocoside and periplocoside aglycone in the preparation of a drug for promoting osteoblast differentiation by increasing the expression of osteoblast-related proteins.

[0018] Preferably, the osteogenesis-related proteins are osteocalcin (OCN), osteoprotegerin (OPG), and type I collagen (Collagen-I, COL-I).

[0019] Preferably, the application is the application of periplocoside and periplocoside aglycone in the preparation of a drug that promotes osteoblast differentiation by activating the BMP-2 (bone morphogenetic protein-2) signaling pathway and upregulating the expression of osteoblast-specific transcription factors.

[0020] The described periwinkle glycosides and periwinkle aglycones can act on sodium / potassium-ATPase (Na + / K +-ATPase, NKA), activates the BMP-2 signaling pathway and upregulates the expression of osteogenic specific transcription factors, promotes osteogenic differentiation and accelerates bone renewal, thereby exerting therapeutic effects on RA. The osteogenic specific transcription factor is Runt-related transcription factor 2 (RUNX2).

[0021] More preferably, the application is that the periwinkle glycosides and periwinkle aglycone are used in the preparation of + / K + -ATPase, NKA) activates the BMP-2 signaling pathway and upregulates the expression of osteoblast-specific transcription factors to promote osteoblast differentiation.

[0022] Another object of the present invention is to use periplocoside and periplocoside aglycone in preparing drugs for treating rheumatoid arthritis.

[0023] Preferably, the application is the use of periplocoside and periplocoside aglycone in the preparation of a drug for treating rheumatoid arthritis by promoting osteoblast differentiation and accelerating bone repair.

[0024] Preferably, the promoting osteoblast differentiation is promoting the differentiation of osteoblast precursors into mature osteoblasts.

[0025] Preferably, the application is the application of periplocoside and periplocoside aglycone in the preparation of a drug for treating rheumatoid arthritis by exerting at least one of the following effects:

[0026] Increase the expression of osteoblast-related proteins to promote osteoblast differentiation; activate the BMP-2 signaling pathway and upregulate the expression of osteoblast-specific transcription factors to promote osteoblast differentiation;

[0027] Reduce the level of inflammatory factors and reduce inflammatory cell infiltration.

[0028] Preferably, the inflammatory factors are TNF-α and IL-6.

[0029] Preferably, the composition comprises an effective therapeutic dose of perilla frutescens glycoside or perilla frutescens aglycone, and pharmaceutically acceptable excipients.

[0030] Another object of the present invention is to provide a drug for treating rheumatoid arthritis, comprising an effective therapeutic dose of periplocoside or periplocoside aglycone, and pharmaceutically acceptable excipients.

[0031] Preferably, the dosage form of the drug is an oral preparation.

[0032] Preferably, the oral preparation is any one of tablets, capsules, granules, suspensions, dripping pills, pills, and oral liquid preparations.

[0033] The tablets are common oral tablets, dispersible tablets, orally disintegrating tablets or sustained-release tablets.

[0034] Beneficial effects of the present invention:

[0035] Periploca glycosides can act on the bone morphogenetic protein-2 (BMP-2) signaling pathway through sodium / potassium-ATPase (NKA), upregulate the expression of osteoblast-related proteins, and thus promote the proliferation and differentiation of osteoblasts. Oral administration of periploca glycosides can dose-dependently alleviate the degree of foot swelling and joint damage in rat RA models, reduce the erosion of joint bone microstructures, and inhibit the levels of multiple inflammatory factors in serum, effectively improving RA symptoms. The present invention provides a new direction for seeking safe, effective and radical RA drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The effects of five representative cardiac glycoside monomers in Achyranthes bidentata on the proliferation and differentiation of MC3T3-E1 cells; A is the effect of five cardiac glycoside monomers on the proliferation of MC3T3-E1 cells; B is the effect of five cardiac glycoside monomers on the differentiation of MC3T3-E1 cells; compared with the negative control group, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001.

[0037] Figure 2 is the effect of periplocoside on the expression of osteogenic related proteins; compared with the negative control group, **** P<0.0001.

[0038] Figure 3 is the effect of periwinkle glycosides on the expression of BMP-2 and RUNX2; compared with the negative control group, * P<0.05, **** P<0.0001.

[0039] Figure 4 NKA is involved in the activation of BMP-2 signaling pathway induced by perilla frutescens; A is the effect of perilla frutescens on NKA activity; B is the sequence of GV248 lentiviral vector elements: hU6-MCS-Ubiquitin-EGFP-IRES-puromycin; C is the mNK1 virus transfection efficiency; D is the effect of NKA on the activation of BMP-2 signaling pathway induced by perilla frutescens; compared with the control group, *p<0.05, ***p<0.001, ****p<0.0001.

[0040] Figure 5The effects of schisandra chinensis on paw swelling and joint scores of CIA rats; A is a typical rat hind paw swelling morphology after 28 days of administration; B is the severity of joint inflammation assessed by paw volume; C is the severity of joint inflammation assessed by joint scores; compared with the model group, * P<0.05, ** P<0.01, *** P < 0.001, **** P<0.0001.

[0041] Figure 6 The effect of periplocoside on osteogenesis; A is HE staining of ankle joint tissue sections of rats in each group; B is OCN immunohistochemical staining of ankle joint tissue sections of rats in each group.

[0042] Figure 7 are three-dimensional reconstruction images of ankle joints; A is a representative Micro-CT image of ankle joints in each group after treatment; B is bone volume fraction (BV / TV); C is bone mineral density (BMD); D is trabecular thickness (Tb.Th); E is trabecular separation (Tb.Sp); compared with the model group, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0043] Figure 8 is the effect of Periploca glycosides on serum inflammatory factors; A is the change of TNF-α content; B is the change of IL-6 content; compared with the model group, ** P<0.01, **** P<0.0001. DETAILED DESCRIPTION

[0044] The technical solution of the present invention is further described below through specific implementation methods.

[0045] Example 1

[0046] Screening of Monomers of Active Cardiac Glycosides from Achyranthes bidentata

[0047] In order to explore the material basis of the anti-RA efficacy of Achyranthes bidentata, the inventors selected 5 representative cardiac glycosides with high content in Achyranthes bidentata according to the literature, namely Periplocin, Periplogenin, Periplocymarin, Digitoxigenin, and Periplogenin3-[O-β-glucopyranosyl-(1→4)-β-sarmentopyranoside. The specific information is shown in Table 1.

[0048] Table 1. Representative cardiac glycosides of Achyranthes bidentata and their structures

[0049]

[0050]

[0051] Mouse embryonic cranial fibroblasts (MC3T3-E1 cells) were used as a source of osteoblasts, and representative cardiac glycosides from Scutellaria baicalensis were screened for their in vitro osteoblast proliferation and differentiation activities.

[0052] Cell culture: Take out the cryopreserved tube containing MC3T3-E1 cells from liquid nitrogen and revive the cells; inoculate the cells into α-MEM complete medium containing 20% ​​FBS, 2mM L-glutamine and 1mM sodium pyruvate, culture them so that the cells are evenly spread on the bottom of the culture flask, place it in a cell culture incubator, and culture it at 37°C, 5% CO2, and 95% relative humidity.

[0053] Drug preparation: Accurately weigh cardiac glycoside monomers (periploca glycoside, periploca aglycone, periploca glycoside, digitoxin aglycone, isoperiploca glycoside) and use dimethyl sulfoxide (DMSO) as solvent to prepare a 1 mM cardiac glycoside monomer mother solution, dilute the mother solution with DMSO to obtain a 100 μM working solution, filter the working solution through a 0.22 μM organic filter membrane for sterilization, and dilute with α-MEM incomplete or complete culture medium to obtain drug-containing culture media of different concentrations (the final concentrations of cardiac glycoside monomers are 100 nM, 20 nM, 5 nM, 1 nM, and 0.1 nM).

[0054] Proliferation activity evaluation: MC3T3-E1 cells in the logarithmic growth phase were collected and cultured at 5×10 3 Cells were inoculated in 96-well plates and cultured overnight in α-MEM complete medium. A control group (Control) and different drug (periplocaine, periplocaine, periplocaine, digitoxin, isoplocaine) treatment groups were set up. The control group was replaced with fresh α-MEM incomplete medium, and the treatment group was replaced with α-MEM incomplete medium containing cardiac glycoside monomers (final concentrations of 0.1, 1, 5, 20, and 100 nM, respectively). After incubation for 48 hours, the medium was replaced with MTT (100 μL / well) with a final concentration of 0.5 mg / mL. After incubation for 4 hours, 100 μL DMSO was added to each well, the absorbance was measured at a wavelength of 570 nm, and the cell survival rate (relative percentage with the Control group) was calculated.

[0055] Evaluation of differentiation-promoting activity: Since alkaline phosphatase (ALP) is highly expressed in osteoblasts and is often used as an indicator of osteogenic differentiation, the ALP enzyme activity test was used to evaluate whether the five representative cardiac glycoside monomers have the activity of promoting osteogenic differentiation of MC3T3-E1 cells. MC3T3-E1 cells were cultured at 3×10 3 Cells were seeded at a density of 100 / well in a 24-well plate and cultured overnight in α-MEM complete medium. A negative control group (Control), a positive control group (osteoblastic induction solution, i.e., α-MEM complete medium containing 50 μg / mL ascorbic acid and 10 mM sodium β-glycerophosphate), and different drug treatment groups (periplocaine, periplocaine, periplocaine, digitoxin aglycone, isoplocaine) were set up. The negative control group was replaced with fresh α-MEM complete medium, the positive control group was replaced with osteoblastic induction solution, and the treatment group was replaced with α-MEM complete medium containing cardiac glycoside monomers (final concentrations of 0.1, 1, 5, 20, and 100 nM, respectively). After 7 days of culture, the culture medium was discarded, PBS was washed twice, and protein lysis buffer without phosphatase inhibitors was added to each well, the cells were collected, centrifuged at 12000g for 15 minutes, and the supernatant was taken. The ALP enzyme activity was determined according to the instructions of the alkaline phosphatase (ALP) kit, and the protein concentration was determined according to the instructions of the BCA kit.

[0056] Results Figure 1 , Table 2 and Table 3 show that: at a concentration of 0.1 to 100 nM, each cardiac glycoside monomer can significantly increase the cell proliferation rate ( Figure 1 A). At specific concentrations, periplocoside, periplocoside aglycone, periplocoside glycoside and digitoxin aglycone can significantly increase the ALP activity of MC3T3-E1 cells ( Figure 1 B). Proliferation and differentiation activities were measured by the Concentration for 50% of maximal effect (EC 50 ) for evaluation. 50 It is an important quantitative indicator of the drug efficacy. 50 The lower the value, the better the drug activity. Among the five representative cardiac glycosides, the EC proliferation promoting effect of periwinkle glycosides was 50 value (0.6nM) and differentiation promoting EC 50 The values ​​(0.98 nM) were all the lowest values ​​(Table 2, Table 3), indicating that periplocoside has a strong ability to promote osteoblast proliferation and differentiation and is an active cardiac glycoside monomer in Scutellaria baicalensis.

[0057] Table 2. EC of representative cardiac glycosides of Achyranthes bidentata on cell proliferation 50 value

[0058]

[0059] Example 2

[0060] Study on the function of periplocin in promoting osteodifferentiation in vitro

[0061] MC3T3-E1 cells were used to further verify the in vitro osteogenic differentiation activity of periploside by detecting osteogenic related proteins. Cell culture was the same as in Example 1.

[0062] Drug preparation: Use DMSO as solvent to prepare a 1 mM stock solution of perilla frutescens; dilute the stock solution with DMSO to obtain a 20 μM working solution, and dilute the working solution with α-MEM complete culture medium to obtain drug-containing culture media of different concentrations (final concentration of perilla frutescens is 20 nM, 5 nM, 1 nM).

[0063] Experimental groups and dosing regimen: MC3T3-E1 cells were cultured at 2×10 4 Cells were inoculated in 6-well plates and cultured at 37°C overnight. A negative control group (Control), a positive control group (osteoblastic induction solution, i.e., α-MEM complete medium containing 50 μg / mL ascorbic acid and 10 mM β-sodium glycerophosphate), and different concentrations of schizonepeta glycoside treatment groups were set up. The negative control group was replaced with fresh α-MEM complete medium, the positive control group was replaced with osteoblastic induction solution, and the treatment group was replaced with α-MEM complete medium containing schizonepeta glycoside (final concentration of 20 nM, 5 nM, 1 nM) for 5 consecutive days. After the culture, the culture medium was discarded, PBS was washed twice, 80 μL of protein lysis solution was added to each well, the cells were collected, centrifuged at 4°C and 12000g for 15 minutes, the supernatant was taken, and Western Blot was used to detect osteoblast-related proteins.

[0064] When performing Western Blot, RIPA lysis buffer containing protease and phosphatase inhibitors was used to separate total cytoplasmic proteins and detected on 4%-20% SDS-PAGE gel. The proteins were transferred to PVDF membranes by constant flow, blocked with 5% skim milk powder, and incubated overnight at 4°C in a buffer containing diluted antibodies. The primary antibody was detected with HRP-conjugated secondary antibodies, and the color was observed on the protein gel imaging system using ECL chemiluminescent liquid, and the protein content was analyzed by density using Image J image analysis software.

[0065] Detection indicators: osteocalcin (OCN), osteoprotegerin (OPG), and type I collagen (Collagen-I, COL-I).

[0066] Results Figure 2, showing that when the concentration of perilla frutescens was 20nM, the relative expression of osteoblast-related protein OCN was significantly increased compared with the negative control group (P<0.0001), and when the concentrations were 1, 5, and 20nM, the relative expression of osteoblast-related proteins OPG and COL-I were significantly increased compared with the negative control group (P<0.0001). Among them, 20nM perilla frutescens and osteogenic induction solution had similar effects on promoting the expression of osteoblast-related proteins, further verifying that perilla frutescens has the ability to induce osteoblast differentiation.

[0067] Example 3

[0068] Study on the mechanism of periplocin in promoting osteodifferentiation in vitro

[0069] In order to explore the intrinsic mechanism of osteogenic differentiation, the inventors detected the changes in the content of key proteins of the BMP-2 signaling pathway in MC3T3-E1 cells after treatment with perilla frutescens.

[0070] The drug preparation, experimental grouping and administration scheme were the same as those in Example 2.

[0071] Detection indicators: bone morphogenetic protein-2 (BMP-2), Runt-related transcription factor 2 (RUNX2).

[0072] Western Blot results are shown in Figure 3 , indicating that: in the concentration range of 1-20nM, periwinkle glycosides can increase the expression of BMP-2 and RUNX2 in a dose-dependent manner, and the effect is most significant at 20nM, while osteogenic induction solution has no significant promoting effect on BMP-2 expression. This result proves that periwinkle glycosides promote osteogenic differentiation by activating BMP-2 and RUNX2.

[0073] Cardiac glycosides are considered to be specific ligands of NKA. Therefore, the inventors studied whether periplocoside can regulate the content and activity of NKA.

[0074] NKA enzyme activity detection: MC3T3-E1 cells were cultured at 5×10 4 Each well was inoculated in a 6-well plate. After overnight culture, the control group (Control) was replaced with α-MEM complete medium, and the treatment group was replaced with a drug-containing medium with a final concentration of 1 nM scutellaria baicalensis glycosides. The culture was continued for 3 days. After the culture was completed, the cells were digested and collected, centrifuged at 1000r for 5 minutes, the supernatant was discarded, and the cell layer was left. 0.2 mL of normal saline was added to each tube to prepare a cell suspension. The protein was extracted by repeated freezing and thawing three times. The enzyme activity was determined according to the instructions of the ultra-micro NKA enzyme test kit, and the protein concentration was determined according to the instructions of the BCA kit. The results of NKA enzyme activity determination are shown in Figure 4A, shows that the administration of perilla frutescens for 3 days can significantly inhibit the activity of NKA enzyme, indicating that NKA is the target of perilla frutescens.

[0075] In order to confirm that NKA is involved in the osteogenic differentiation process induced by the BMP-2 signaling pathway, the inventors constructed a MC3T3-E1 cell model with low NKA expression by lentiviral transfection. The sequence of lentiviral vector elements is shown in Figure 4 B. Construction of MC3T3-E1 cell line with low NKA expression: With the help of Shanghai Jikai Gene Chemistry Technology Co., Ltd., the inventors designed and synthesized a small interfering RNA (siRNA) sequence (5′-CAGTAACATTCCGGAAATC-3′) based on the characteristics of the mouse NKAα1 subunit mRNA sequence, and synthesized the corresponding oligonucleotide sequence based on this, cloned it into the GV248 plasmid, and finally successfully packaged and obtained siRNA lentiviral particles that can encode the mRNA targeting NKA α1 subunit, which is used to silence the expression of mouse NKAα1 subunit and named mNK1 virus. MC3T3-E1 cells were inoculated in 6-well plates at a density of 6×10 4 cells / ml, and cultured in α-MEM complete medium for 24 h, then added infection enhancer HiTransG P and mNK1 virus with a multiplicity of infection (MOI) of 100. After incubation for 16 h, the complete medium was replaced and cultured for another 72 h. Successful expression of lentivirus was observed under a fluorescence microscope ( Figure 4 C). The cells were passaged to two 6-well plates, and the α-MEM complete medium and the drug-containing medium with a final concentration of 20 nM perilla frutescens were replaced respectively. After culturing for 3 days, proteins were extracted for Western Blot analysis.

[0076] Detection indicators: NKA, BMP-2, RUNX2.

[0077] Western Blot results showed that in MC3T3-E1 cells transfected with mNK1 virus, the expression of NKA was effectively silenced (P<0.0001); in cells with normal NKA expression, periwinkle glycosides could significantly increase the expression of BMP-2 and RUNX2 (P<0.0001); in cells with a large amount of NKA knocked down, periwinkle glycosides could not effectively activate BMP-2, and the expression of downstream RUNX2 was also significantly inhibited ( Figure 4 D). Therefore, NKA participates in mediating the activation of BMP-2 signaling pathway induced by periplocin, thereby promoting osteogenic differentiation.

[0078] Example 4

[0079] Effects of Periploca glycosides on collagen-induced bone destruction in rheumatoid arthritis

[0080] The collagen-induced rheumatoid arthritis (CIA) rat model was used to evaluate the anti-RA activity of periplocoside in vivo.

[0081] Preparation of modeling agent: Add bovine type II collagen solution (CII, 2 mg / mL) dropwise into an equal volume of complete Freund's adjuvant (CFA, 2 mg / mL), vortex until uniform, and make sure that the solution does not diffuse when dropped into water, to obtain a bovine type II collagen emulsion with a final concentration of 1 mg / mL. The whole process must be performed on ice.

[0082] Model establishment: Rats were anesthetized by intraperitoneal injection of 2% (w / v) sodium pentobarbital solution (0.2 mL / 100 g), and the base of their tails were disinfected. Except for the control group, rats in each group were intradermally injected with 100 μL of bovine type II collagen emulsion at multiple points at the base of the tail (i.e., the injection volume of bovine type II collagen was 100 μg). One week later, a booster immunization was performed at the same site with the same dose.

[0083] Experimental groups: Forty-eight Wistar female rats weighing 160-180 g were randomly divided into 6 groups, namely: control group (Con), model group (Mod), low-dose periploside group (G-2), high-dose periploside group (G-4), chemical positive drug group (MTX) and traditional Chinese medicine positive drug group (Capsule), with 8 rats in each group.

[0084] Dosage regimen: The drug is administered by gavage for 28 consecutive days. The dosage and frequency are as follows:

[0085] Control group (Con): 0.5% CMC-Na solution, once a day;

[0086] Model group (Mod): 0.5% CMC-Na solution, once a day;

[0087] Low-dose periploside group (G-2): 2 mg / kg periploside (using 0.5% CMC-Na as solvent to make a uniform suspension), once a day;

[0088] High-dose periploside group (G-4): 4 mg / kg periploside (using 0.5% CMC-Na as solvent to make a uniform suspension), once a day;

[0089] Chemical positive drug group (MTX): 0.5 mg / kg methotrexate (using 0.5% CMC-Na as solvent to make a uniform solution); 2 times / week;

[0090] Chinese medicine positive group (Capsule): 0.25 g / kg content of Heibuteng Zhuifeng Huoluo capsule (using 0.5% CMC-Na as solvent to make a uniform suspension), once a day.

[0091] Ankle swelling measurement and arthritis score of CIA rats: After the model was established, the paw volume of CIA rats was measured every 4 days using a toe volume meter to observe the inhibitory effect of drugs on the swelling of the paws of CIA rats. The severity of arthritis was evaluated using a 5-level scoring method, and the arthritis index standard was as follows: 0 = no swelling, focal redness; 1 = swelling of the finger joints; 2 = mild swelling of the ankle or wrist; 3 = severe inflammation involving the entire paw; 4 = joint deformity or stiffness. The sum of the arthritis indexes of the two hind limbs was recorded, and the arthritis index = 6-8 was used as an indicator of successful model construction.

[0092] Staining and immunohistochemical analysis of ankle joint tissues of CIA rats: On the 28th day after administration, all rats were killed by cervical dislocation after blood sampling, and the ankle joints of the right hind limbs were separated, fixed with 4% paraformaldehyde, decalcified with 10% EDTA, routinely dehydrated, embedded in paraffin, and sectioned.

[0093] The tissues were stained with hematoxylin and eosin (H&E) and the histopathological differences were observed under an optical microscope. At the same time, immunohistochemical staining was performed on the sections with OCN as the detection object to analyze the expression level of OCN.

[0094] Determination of bone density and bone morphological parameters: Three left hind limb ankle joints were randomly selected from each group, fixed with 4% paraformaldehyde, and then subjected to Micro-CT scanning imaging to obtain three-dimensional reconstruction images of the ankle joints. The following three-dimensional parameters were analyzed and calculated: bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Bone volume fraction (BV / TV) reflects the trabecular bone mass of different samples. When bone anabolism is greater than catabolism, this value increases and bone mass increases; bone density (BMD) is used to measure bone mass, and an increase in this value indicates an increase in bone mass; trabecular thickness (Tb.Th) and separation (Tb.Sp) are the main indicators for evaluating the spatial morphological structure of trabecular bone. When bone anabolism is greater than bone catabolism, Tb.Th increases and Tb.Sp decreases.

[0095] Enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of serum TNF-α and IL-6.

[0096] The results of toe swelling and arthritis score showed that compared with the control group, the toes of the model group rats were significantly swollen, and the arthritis index was maintained at 6-8, indicating that the rat RA model was successfully established; oral administration of scutellaria baicalensis glycosides (2, 4 mg / kg) can dose-dependently alleviate the swelling of the ankle joints and soles of the hind limbs of rats induced by CII-CFA, and significantly reduce the arthritis index. The effect of high-dose scutellaria baicalensis glycosides is comparable to that of methotrexate, and is superior to that of Heibuteng Zhuifeng Huoluo Capsule ( Figure 5 ).

[0097] Pathological and immunohistochemical results showed that the cells in the control group were arranged evenly and neatly, the cell nuclei were clearly visible, and there was no synovial thickening and angiogenesis; while the cells in the model group were arranged disorderly, the synovial membrane showed inflammatory cell infiltration and hyperplasia, some cell nuclei were necrotic, and the expression of OCN was significantly reduced; after the administration of periploside, the synovial membrane of the joint gradually returned to its normal physiological state, the inflammatory cells decreased, and the expression of OCN was adjusted back ( Figure 6 A, B).

[0098] Micro-CT scanning reconstruction showed that compared with the control group, the surface of the ankle and toe joint bones of the rats in the model group were loose and rough, uneven, and the bone structure was severely eroded; compared with the model group, periplocoside could improve the degree of joint bone destruction in CIA rats to a certain extent, and gradually restore the bone structure of the toe joint to a normal physiological state ( Figure 7 A). At the same time, compared with the control group, the BV / TV, BMD, and Tb.Th of the model group were all decreased, and Tb.Sp was increased, indicating that the model was successfully established; the BV / TV, BMD, and Tb.Th of the low-dose and high-dose schizonepeta group and the two positive drug groups were all increased, and Tb.Sp was decreased, indicating that bone anabolism was higher than catabolism after drug administration, bone mass increased, and RA symptoms were alleviated ( Figure 7 A- Figure 7 E). This indicates that periplocoside has the effect of accelerating bone formation and promoting bone reconstruction, and can be used to treat bone damage caused by RA.

[0099] Compared with the control group, the levels of inflammatory factors TNF-α and IL-6 in the serum of the model group rats were significantly increased, while periwinkle glycosides could significantly reduce the levels of inflammatory factors TNF-α and IL-6 in the serum ( Figure 8 ).

[0100] In summary, periplocoside has good bone-promoting activity in vitro and in vivo, can effectively improve bone loss and damage caused by RA, and can be further used in the preparation of drugs for the treatment of RA.

Claims

1. Application of periplocoside and periplocoside aglycone in the preparation of drugs that promote osteocyte proliferation.

2. Application of periplocoside and periplocoside aglycone in the preparation of drugs that promote osteoblast differentiation.

3. The use according to claim 2, characterized in that: The application is the application of periplocoside and periplocoside aglycone in the preparation of drugs for promoting osteoblast differentiation by increasing the expression of osteoblast-related proteins; the osteoblast-related proteins are osteocalcin, osteoprotegerin and type I collagen.

4. The use according to claim 2, characterized in that: The application is the application of periplocoside and periplocoside aglycone in the preparation of a drug that promotes osteoblast differentiation by activating the BMP-2 signaling pathway and upregulating the expression of osteoblast-specific transcription factors; the osteoblast-specific transcription factor is Runt-related transcription factor 2.

5. Application of periplocoside and periplocoside aglycone in the preparation of drugs for the treatment of rheumatoid arthritis.

6. The use according to claim 5, characterized in that: The application is the application of periplocoside and periplocoside aglycone in the preparation of a drug for treating rheumatoid arthritis by promoting cell osteogenic differentiation and accelerating bone repair.

7. The use according to claim 5, characterized in that: The application is the application of periplocoside and periplocoside aglycone in the preparation of a drug for treating rheumatoid arthritis by exerting at least one of the following effects: Increase the expression of osteoblast-related proteins to promote osteoblast differentiation; activate the BMP-2 signaling pathway and upregulate the expression of osteoblast-specific transcription factors to promote osteoblast differentiation; Reduce the level of inflammatory factors and reduce inflammatory cell infiltration.

8. A drug for treating rheumatoid arthritis, characterized in that: It contains an effective therapeutic dose of perilla frutescens glycoside or perilla frutescens aglycone, and pharmaceutically acceptable excipients.

9. The drug according to claim 8, characterized in that: The medicine is an oral preparation.

10. The drug according to claim 9, characterized in that: The oral preparations are tablets, capsules, granules, suspensions, dripping pills, pills, and oral liquid preparations.

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