WO2021249420A1 - 血筒素在制备抗类风湿关节炎药物中的应用 - Google Patents

血筒素在制备抗类风湿关节炎药物中的应用 Download PDF

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WO2021249420A1
WO2021249420A1 PCT/CN2021/099072 CN2021099072W WO2021249420A1 WO 2021249420 A1 WO2021249420 A1 WO 2021249420A1 CN 2021099072 W CN2021099072 W CN 2021099072W WO 2021249420 A1 WO2021249420 A1 WO 2021249420A1
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rats
group
rheumatoid arthritis
cells
blood
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French (fr)
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王炜
余黄合
刘斌
蔡雄
李斌
彭彩云
江星明
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湖南中医药大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]

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  • the present invention relates to a botanical medicine, in particular to the application of blood tubin in the preparation of an anti-rheumatoid arthritis medicine.
  • the blood tube is a commonly used medicinal plant of the Tujia nationality. It belongs to the Schisandra genus Kadsura heteroclita (Roxb) Craib of the Schisandra family. Its cane is often used as medicine.
  • the blood tube is sweet in nature, slightly pungent and warm. It has the effects of nourishing blood and promoting blood circulation, expelling wind and dampness, promoting qi and relieving pain.
  • Tujia people are often used to treat rheumatic arthralgia, epigastric pain, menstrual pain, bone pain, rheumatoid arthritis, lumbar muscle strain, colds, postpartum rheumatic paralysis and other diseases.
  • the 2005 edition of the Chinese Pharmacopoeia is included as "Hetero-shaped South Schisandra cane", which is one of the main ingredients of Zhonghua Dieda Pills, which is used to treat traumatic injuries.
  • Schisanlactone E is a triterpenoid compound pentalactone E (schisanlactone E) extracted from the blood cylinder.
  • Existing research believes that it has anti-tumor effects.
  • Rheumatoid arthritis (rheumatoidarthritis, RA) is a chronic, systemic disease of unknown etiology, mainly synovial inflammatory hyperplasia. It is characterized by multiple joints, symmetry, and aggressive joint inflammation of the hand and foot facet joints, often accompanied by involvement of extra-articular organs and positive serum CRPL and rheumatoid factor, which can lead to joint deformities and loss of function, leading to loss of labor or cause Disabled.
  • the pathological features of RA mainly include the proliferation of synovial lining cells, the infiltration of a large number of inflammatory cells in the interstitium, the neovascularization of microvessels, the formation of pannus, and the destruction of cartilage and bone tissue.
  • rheumatoid arthritis There is no cure for rheumatoid arthritis.
  • the main purpose of current treatment is to reduce joint inflammation, inhibit histopathological changes and bone destruction, protect joint and muscle function as much as possible, and finally achieve complete remission or reduced disease activity.
  • Target the commonly used drugs for the treatment of RA are indomethacin and other non-steroidal anti-inflammatory drugs and methotrexate and other anti-tumor drugs, but these drugs have relatively large side effects, and long-term use will produce strong adverse reactions.
  • the technical problem to be solved by the present invention is to provide a botanical medicine for treating RA, provide a botanical component that can be used for a long time for RA patients, and enrich RA treatment methods.
  • the present invention provides the application of spheroidin in the preparation of anti-rheumatoid arthritis drugs.
  • the spheroidin has the structure shown in the following formula (I):
  • the present invention also provides an anti-rheumatoid arthritis medicine, the anti-rheumatoid arthritis medicine uses hemagglutinin as the main active ingredient.
  • the anti-rheumatoid arthritis medicine also contains pharmaceutically acceptable salts, esters, sugars or excipients.
  • the anti-rheumatoid arthritis drug can be a tablet, a capsule or an injection.
  • hemostatin has a significant effect of inhibiting the viability of rheumatoid arthritis fibroblast-like synovial cells, inhibiting the inflammatory factors produced by rheumatoid arthritis fibroblast-like synovial cells, and can inhibit macrophages.
  • the cells produce inflammatory factors, inhibit the infiltration of inflammatory cells and synovial hyperplasia, improve the symptoms of rheumatoid arthritis, and are safe to use.
  • Cystin provides a plant raw material drug with a significant anti-rheumatoid arthritis effect, which can be used in anti-rheumatoid arthritis drugs and can be made into a variety of pharmaceutical dosage forms.
  • Figure 1 is a comparison of the effects of SE and common anti-RA drugs on the viability of RAFLS cells
  • Figure 2 is a comparison of the IC 50 value of SE and common anti-RA drugs on the viability of RAFLS cells
  • Figure 3 is a comparison of the effects of SE and common anti-RA drugs on the TNF- ⁇ produced by RAFLS;
  • Figure 4 is a comparison of the effects of SE and common anti-RA drugs on the IL-6 produced by RAFLS;
  • Figure 5 is a comparison of the effects of SE and common anti-RA drugs on the IL-1 ⁇ produced by RAFLS;
  • Figure 6 is a comparison of the effects of SE and common anti-RA drugs on the IL-10 produced by RAFLS;
  • Figure 7 is a comparison of the effects of SE and commonly used anti-RA drugs on LPS-induced TNF- ⁇ produced by mouse RAW264.7 macrophages;
  • Figure 8 is a comparison of the effects of SE and common anti-RA drugs on the IL-6 produced by LPS-induced mouse RAW264.7 macrophages;
  • Figure 9 is a comparison of the effects of SE and commonly used anti-RA drugs on the IL-1 ⁇ produced by LPS-induced mouse RAW264.7 macrophages;
  • Figure 10 is a comparison of the effects of SE and common anti-RA drugs on the IL-10 produced by LPS-induced mouse RAW264.7 macrophages;
  • Figure 11 is a comparison of the hemolysis rate of SE whole blood at different concentrations
  • Figure 12 is a comparison of the effects of SE and common anti-RA drugs on the organs and tissues of AIA rats;
  • Figure 13 is a comparison of the effects of SE and common anti-RA drugs on the body weight of AIA rats;
  • Figure 14 is a comparison of the effects of SE and common anti-RA drugs on toe swelling in AIA rats;
  • Figure 15 is a comparison of SE and commonly used anti-RA drugs on toe swelling in AIA rats;
  • Figure 16 is a comparison of the effects of SE and common anti-RA drugs on the arthritis index of AIA rats;
  • Figure 17 is a comparison of the effects of different doses of SE on the pathological tissues of the toes of AIA rats;
  • Figure 18 is a comparison of the effects of different doses of SE on the pathological scores of the toes of AIA rats;
  • Figure 19 is a CT image comparison of the effects of different doses of SE on the toe bones of AIA rats;
  • Figure 20 is a comparison of the effects of different doses of SE on TNF- ⁇ in the serum of AIA rats;
  • Figure 21 is a comparison of the effects of different doses of SE on IL-6 in the serum of AIA rats;
  • Figure 22 is a comparison of the effects of different doses of SE on IL-1 ⁇ in the serum of AIA rats;
  • Figure 23 is a comparison of the effects of different doses of SE on IL-17 in the serum of AIA rats;
  • Figure 24 is a comparison of the effects of different doses of SE on IL-23 in the serum of AIA rats;
  • Figure 25 is a comparison of the effects of different doses of SE on ALT in the blood of AIA rats;
  • Figure 26 is a comparison of the effects of different doses of SE on the AST in the blood of AIA rats;
  • Figure 27 is a comparison of the effects of different doses of SE on ALP in the blood of AIA rats;
  • Figure 28 is a comparison of the effects of different doses of SE on CREA in the blood of AIA rats;
  • Figure 29 is a comparison of the effects of different doses of SE on the WBC in the blood of AIA rats;
  • Figure 30 is a comparison of the effects of different doses of SE on the CPR of AIA rat blood
  • Figure 31 is a comparison of the effects of different doses of SE on the blood MONO of AIA rats;
  • Figure 32 is a comparison of the effects of different doses of SE on the blood RBC of AIA rats;
  • Figure 33 is a comparison of the effects of different doses of SE on the MCV in the blood of AIA rats;
  • Figure 34 is a comparison of the effects of different doses of SE on the HCT of AIA rat blood
  • Figure 35 is a comparison of the effects of different doses of SE on PLT in the blood of AIA rats;
  • Figure 36 is a comparison of the effects of different doses of SE on HGB in the blood of AIA rats;
  • Figure 37 is a comparison of the effects of different doses of SE on WBC in the blood of AIA rats.
  • RAFLS cells were purchased from Beijing Beina Chuanglian Biotechnology Research Institute; mouse RAW264.7 macrophages were purchased from Xiangya Cell Bank of Central South University; SPF SD rats were purchased from Slack, Hunan province Jingda Laboratory Animal Co., Ltd. (certificate number: 43004700063752).
  • High-sugar DMEM medium was purchased from Gibco, USA; 1 ⁇ PBS was purchased from Gibco, USA; double antibody Penicillin-Streptomycin was purchased from Gibco, USA; pancreatin cell digestion solution (containing 0.25% pancreatin ( Trypsin) and 0.02% EDTA, without Ca2+ and Mg2+) were purchased from Gibco, USA; Fetal bovine serum was purchased from Gibco, USA; MTT cell proliferation and cytotoxicity test kit was purchased from Shanghai Biyuntian Biotechnology Co., Ltd.; Indomethacin Xin (content ⁇ 99.0%) was purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; TNF- ⁇ , IL-6, IL-1 ⁇ ELISA kits were purchased from Wuhan Boster Biotechnology Co., Ltd.; heat quenching Live Mycobacterium tuberculosis H37Ra (Sigma Aldrich, USA, 20150411); Mineral oil (Sigma Aldrich, USA,
  • the instruments and instruments used are German Menet vernier calipers (Shanghai Menet Industrial Co., Ltd., model MNT-150); high-resolution small animal micro-CT (U.S. PerkinElmer-Caliper LS Quantum FX Demo); high-speed refrigerated centrifuge ( Eppendorf 5810R, Germany; Tissue sample grinder (IKA A11, Germany); Homogenizer (IKA T10basic, Germany); Gas anesthesia machine (SurgiVetCDS9000); Electronic balance (Shimadzu ATY224, Japan); Electric heating Blow drying oven (Tianjin Test Instrument Co., Ltd., model: 101-1AB); ice maker (SCOTSMAN, model: AF100); nucleic acid protein expression imaging system (Bio-Rad ChemiDoc XRS+). Others are common commercial products.
  • RAFLS rheumatoid arthritis fibroblast-like synovial cells
  • RAFLS cell culture uses 100U/ml penicillin, 100 ⁇ g/ml streptomycin as double antibodies, 10% fetal bovine serum (FBS) in DMEM/F-12 medium for a total of 7mL, placed in 6cm cells In a petri dish, culture in a 37°C, 5% CO 2 incubator. After the cells adhere to the wall and grow well, pass them once every 2-3 days.
  • FBS fetal bovine serum
  • RAFLS cells Take the RAFLS cells in the logarithmic growth phase in a 6cm cell culture dish and wash them twice with 1 ⁇ PBS. Take 500 ⁇ L of 0.25% trypsin cell digestion solution and place it in a 37°C incubator for 4 minutes, then use 1mL of 10% FBS.
  • DMEM medium was used to terminate the digestion, the cells were collected, placed in a 1.5mL EP tube, centrifuged at 900 rpm for 5 minutes, the supernatant was removed, and then 1 mL of 10% FBS-containing DMEM/F-12 medium was added to prepare a single cell suspension, and then 10 mL containing 10% FBS-containing DMEM/F-12 medium was made into 1 ⁇ 105 cells/mL single cell suspension, and 100 ⁇ L per well was uniformly seeded in a 96-well plate, and placed in a 37°C, 5% CO 2 incubator for culture.
  • DMSO DMSO 100 ⁇ L
  • the cell viability of the drug-administered group (OD value of the drug-administered group/OD value of the blank control group) ⁇ 100% was used to calculate the cell viability of the drug-administered group.
  • RAFLS cells Take the RAFLS cells in the logarithmic growth phase in a 6cm cell culture dish and wash them twice with 1 ⁇ PBS. Take 500 ⁇ L of 0.25% trypsin digestion solution and place them in a 37°C incubator for 4 minutes, and then use 1mL of 10% FBS DMEM /F-12 medium to terminate the digestion, collect the cells, place them in a 1.5mL EP tube, centrifuge at 900 rpm for 5 minutes, remove the supernatant, and then add 1 mL of 10% FBS DMEM/F-12 medium to prepare a single cell suspension, and then Use 10 mL of DMEM medium containing 10% FBS to prepare a 1 ⁇ 10 5 cell/mL single cell suspension, inoculate 100 ⁇ L per well in a 96-well plate evenly, and place it in a 37° C., 5% CO 2 incubator for culture.
  • each drug has 6 complexes for each concentration. hole. After culturing in a 37°C, 5% CO 2 incubator for 48 hours, discard the medium and observe the cell growth status. After incubating for 4 hours with a serum-free medium containing 10% MTT, discard the supernatant and add 100 ⁇ L of DMSO to each well.
  • the inhibitory effects of different concentrations of SE, methotrexate, indomethacin and sinomenine on the cell viability of RAFLSs are shown in Figure 2.
  • a The IC 50 values of methotrexate, indomethacin and sinomenine on the viability of RAFLSs cells are shown in Table 1. It can be seen that the IC 50 values of SE, methotrexate, indomethacin, and sinomenine on the viability of RAFLS cells are 9.12, 2.96, 3.81, 62.87 ⁇ M, respectively.
  • SE can effectively inhibit the proliferation and growth of RAFLS, and therefore can inhibit synovial hyperplasia during the occurrence of rheumatoid arthritis.
  • RAFLS cell culture uses 100U/ml penicillin, 100 ⁇ g/ml streptomycin as double antibodies, 10% fetal bovine serum (FBS) in DMEM/F-12 medium for a total of 7mL, placed in 6cm cells In a petri dish, culture in a 37°C, 5% CO 2 incubator. After the cells adhere to the wall and grow well, pass them once every 2-3 days.
  • FBS fetal bovine serum
  • RAFLS cells Take the RAFLS cells in the logarithmic growth phase from a 6cm cell culture dish and wash them twice with 1 ⁇ PBS. Take 500 ⁇ L of 0.25% trypsin digestion solution and place them in a 37°C incubator for 4 minutes, and then use 1mL 10% FBS DMEM medium to terminate Digest, collect the cells, place them in a 1.5mL EP tube, centrifuge at 900 rpm for 5 min, remove the supernatant, and add 1 mL of 10% FBS in DMEM/F-12 medium to prepare a single cell suspension, and then use 10 mL of 10% FBS containing DMEM medium was made into 1 ⁇ 10 5 cells/mL single cell suspension, and 100 ⁇ L per well was uniformly inoculated in a 96-well plate, and cultured in a 37°C, 5% CO 2 incubator.
  • indomethacin at a concentration of 5.0 ⁇ M can significantly reduce the expression levels of inflammatory factors TNF- ⁇ , IL-6, and IL-1 ⁇ in RAFLS (*P ⁇ 0.05 or **P ⁇ 0.01) , And significantly increase the expression level of IL-10 (*P ⁇ 0.05 or **P ⁇ 0.01).
  • SE stimulated RAFLS cells at concentrations of 4.5, 9.0, and 18.0 ⁇ M for 24 hours, and they all significantly inhibited the expression of the inflammatory factors TNF- ⁇ , IL-6, and IL-1 ⁇ secreted by them (*P ⁇ 0.05 Or **P ⁇ 0.01), and increase the expression level of IL-10 (*P ⁇ 0.05 or **P ⁇ 0.01).
  • Mouse RAW264.7 macrophages use 100U/ml penicillin, 100 ⁇ g/mL streptomycin as double antibodies, 10% fetal bovine serum (FBS) high-glycemic DMEM medium, totaling 7mL, and place them in a 6cm cell culture dish In the medium, culture in a 37°C, 5% CO 2 incubator. After the cells adhere to the wall and grow well, pass them once every 2-3 days.
  • FBS fetal bovine serum
  • indomethacin at a concentration of 5 ⁇ M can significantly reduce the expression levels of inflammatory factors TNF- ⁇ , IL-6, and IL-1 ⁇ in RAFLS (*P ⁇ 0.05 or **P ⁇ 0.01), And significantly increase the expression level of IL-10 (*P ⁇ 0.05 or **P ⁇ 0.01).
  • SE stimulated RAFLS cells at concentrations of 4.5, 9.0, and 18.0 ⁇ M for 24 hours, and they all significantly inhibited the expression of the inflammatory factors TNF- ⁇ , IL-6, and IL-1 ⁇ secreted by them (*P ⁇ 0.05 Or **P ⁇ 0.01), and increase the expression level of IL-10 (*P ⁇ 0.05 or **P ⁇ 0.01).
  • SE can effectively inhibit the inflammatory factors produced by RAFLS and mouse RAW264.7 macrophages, and increase the expression level of IL-10, so it can inhibit inflammation during the occurrence of rheumatoid arthritis.
  • the production of sexual response reduces the symptoms of rheumatoid arthritis and the damage to the body.
  • AIA cystin on adjuvant arthritis
  • the rat whole blood sample was centrifuged at 3000 rpm at 4°C for 5 min, and washed with PBS 5 times to collect pure red blood cells.
  • Combine 50 ⁇ L of 4% red blood cells (v/v) with 950 ⁇ L of SE with different concentrations (0.21, 0.42, 0.84, 1.68 and 3.36mg/mL) (dispersants are PBS with pH 7.4), and 50 ⁇ L of 4% red blood cells (v/v) It was mixed with 950 ⁇ L of PBS, and the resulting mixture was incubated at 37°C for 4h.
  • the positive control uses red blood cells mixed with pure water as 100% hemolysis.
  • Hemolysis(%) (I/I 0 ) ⁇ 100% to calculate the percentage of hemolysis.
  • Hemolysis represents the hemolysis rate
  • I represents the absorbance of the supernatant after adding different concentrations of SE to the red blood cell suspension
  • I 0 represents the absorbance of complete hemolysis in pure water. All hemolysis experiments were repeated three times.
  • the rat red blood cells were completely hemolyzed in pure water, with a hemolysis rate of 100%, and no hemolysis occurred in 1 ⁇ PBS, with a hemolysis rate of 0%.
  • the hemolysis rate of red blood cells under the conditions of SE concentration of 0.21, 0.42, 0.84, 1.68, and 3.36 mg/mL were 1.04, 1.05, 1.06, 1.41, 1.45%, respectively. It can be seen that SE meets the ISO standard of hemolysis rate ⁇ 5%, and meets the safety standard for use as a medicine.
  • the rats were randomly divided into 6 groups (6 rats in each group), namely: blank group (Normal), model group (Model), indomethacin group (Indo 1.0 mg/kg), SE low-dose group (SE 0.21mg/kg), SE medium-dose group (SE 0.42mg/kg), SE high-dose group (SE 0.84mg/kg).
  • the rats in each group were collected blood from the orbit, centrifuged at 4°C and 3500 rpm for 15 minutes, and the upper serum was separated and stored in the refrigerator at -80°C for later use.
  • the rats were anesthetized with a small animal gas anesthesia machine, and the hindfoot joints of each group of rats were scanned with small animal CT to evaluate the degree of joint swelling and bone destruction.
  • the rats were anesthetized, and the blood was collected by abdominal aorta blood sampling. The blood was centrifuged at 4°C and 3500 rpm for 15 minutes. A part of the whole blood of the obtained rat was taken and analyzed by an automatic blood routine tester.
  • the blood routine indexes of each rat in the group, and the other part of the blood separated upper serum was used for the determination of blood biochemical indexes such as ALT, AST and inflammatory factors.
  • the rats were euthanized and the main organs (heart, liver, spleen, lung and kidney) were collected, and toes were cut from above the ankle joint, half of each were fixed with 4% paraformaldehyde, and the other part was frozen at -80°C Save for use in protein extraction.
  • the model group As shown in Figure 13, compared with the blank group of rats, from the 9th day after the AIA model, the model group has decreased appetite, severe hindfoot swelling, inconvenience, reduced activity, and poor flexibility, etc. Arthritis symptoms, joint deformities, slow weight gain, and in the severe period of arthritis, that is, the weight gain of rats stagnates and shows a downward trend on the 9th to 15th days. After the indomethacin group was given indomethacin 1.0 mg/kg treatment, the quality of life of the rats was significantly improved. After each SE dose group was given SE 0.21, 0.42, 0.84mg/kg, the weight of the rats maintained a growth trend.
  • the incidence rate of the model group is 100% and the incidence is the most severe.
  • the incidence rate of rats in the SE 0.42 mg/kg dose group is 75%, and the SE 0.84 mg/kg dose group is 20%, the severity of the disease was reduced in a dose-dependent manner. From the measured thickness of toe swelling in rats, the toe swelling of rats in the indomethacin group was not obvious after indomethacin treatment.
  • SE can significantly reduce the AIA model at doses of 0.21, 0.42, and 0.84 mg/kg The toes of rats were swollen, and the arthritis index was reduced in a dose-dependent manner. The difference was statistically significant (*P ⁇ 0.05, **P ⁇ 0.01 or ***P ⁇ 0.001).
  • rat hind toes fixed with formalin were decalcified, embedded in paraffin, and sectioned and stained with hematoxylin-eosin (HE). Observed under an optical microscope at a magnification of 10 ⁇ 40. The degree of joint cavity narrowing, synovial tissue hyperplasia, inflammatory cell infiltration, and pannus hyperplasia were scored pathologically.
  • the scoring method is: normal joint space, no obvious synovial tissue hyperplasia, no inflammatory cell infiltration, no pannus neoplasia pathology all get 0 points; joint space is slightly narrow, slight synovial tissue hyperplasia, a few inflammatory cells Infiltration and very slight pannus neoplasia are scored 1 point; joint space stenosis, moderate synovial tissue hyperplasia, a large number of inflammatory cell infiltration, and mild pannus neoplasia are scored 2 points; joint space is obviously narrowed and severe Synovial tissue hyperplasia, a large number of inflammatory cell infiltration, and severe pannus neoplasia were scored 3 points.
  • the CT image on the 21st day after modeling is shown in Figure 19. It can be seen that the interphalangeal joints and metatarsophalangeal joints of the blank group have a good corresponding relationship, the joint space is clear, no narrowing or widening is seen, and the articular surface is smooth.
  • rats in the model group showed severe bone erosions, a large number of osteophytes proliferated, joint cavities narrowed or even disappeared, and joints were severely deformed.
  • rats in the indomethacin group and the SE medium and high-dose groups had milder posterior toe joint bone erosion and joint space stenosis, and the joint structure was relatively complete. Better protection.
  • the serum obtained after centrifugation of rat whole blood was used to detect the expression levels of TNF- ⁇ , IL-6, IL-1 ⁇ , IL-17, IL-23 and other inflammatory factors in the serum with Elisa kit.
  • the test results are shown in Figure 20-24.
  • the serum levels of TNF- ⁇ , IL-6, IL-1 ⁇ , IL-17, IL-23 and other inflammatory factors in the model group were significantly increased ( # P ⁇ 0.05), compared with the model group, the medium and high dose (0.42mg/kg and 0.84mg/kg) groups can significantly down-regulate TNF- ⁇ , IL-6, IL-1 ⁇ , IL-17, IL -23 inflammatory factor level (*P ⁇ 0.05 or **P ⁇ 0.01), SE low dose (0.21mg/kg) can significantly reduce the expression level of TNF- ⁇ , IL-6, IL-17, and IL-23 ( *P ⁇ 0.05 or **P ⁇ 0.01).
  • the indomethacin group can significantly inhibit the expression of TNF- ⁇ , IL-6, IL-1 ⁇ , IL-17, and IL-23 (*P ⁇ 0.05 or **P ⁇ 0.01). It is suggested that SE may exert anti-AIA pharmacological effects by inhibiting the expression of key inflammatory cytokines.
  • the blood biochemical test results of rat serum are shown in Figure 25-28.
  • the blood ALT, AST, ALP, and CREA of the model group increased significantly, indicating that the adjuvant has a greater effect on arthritis.
  • the liver and kidney function of mice has certain damage ( # P ⁇ 0.05).
  • each dose group of SE (0.21, 0.42, 0.84mg/kg) can significantly reduce the content of ALT, AST, ALP, CREA in the blood of AIA model rats (*P ⁇ 0.05 or **P ⁇ 0.01). It shows that SE protects the liver and kidney tissues of rats during the treatment of arthritis.
  • the results of blood routine analysis of blood cells are shown in Figure 29- Figure 31.
  • the model group has WBC, C-reactive protein level (CRPL), and monocyte (MONO).
  • CRPL C-reactive protein level
  • MONO monocyte
  • the proportion of inflammatory cells in the blood of rats in the positive drug indomethacin treatment group was significantly reduced.
  • hematocin can effectively inhibit the proliferation of fibroblast-like synovial cells in rheumatoid arthritis, and effectively inhibit the inflammatory factors TNF- ⁇ and IL-1 ⁇ of RAFLS induced by lipopolysaccharide (LPS). , IL-6 production, and increase the expression level of IL-10. It can also effectively inhibit the production of inflammatory factors TNF- ⁇ , IL-1 ⁇ , and IL-6 in RAW264.7 induced by LPS, and increase the expression level of IL-10, showing its anti-inflammatory effect.
  • LPS lipopolysaccharide
  • SE Radiological observation of rat toe joints by small animal CT scans showed that SE significantly improved the erosion of rat’s hind toe joint bone and cartilage, reduced the degree of joint space stenosis, and protected the joint structure tended to be intact under each dose of SE. SE has a good protective effect on the toe joints of rats. The results show that SE can be used as a drug for the treatment of rheumatoid arthritis, providing a new, clear-cut and safe plant-derived drug for the treatment of rheumatoid arthritis.

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Abstract

涉及植物药，公开了血筒素在制备抗类风湿关节炎药物中的应用。血筒素能有效的抑制类风湿关节炎成纤维样滑膜细胞(RAFLS)增生，有效抑制脂多糖诱导的RAFLS的炎性因子TNF-α、IL-1β、IL-6的产生，升高IL-10的表达水平，具有较好的抗炎作用。并能有效的抑制AIA诱导的大鼠关节炎的发生率，降低大鼠足趾肿胀度，改善大鼠足趾的病理和放射学影像上的变化。还公开了一种以血筒素为活性成分的药物，可制成片剂、胶囊剂或者针剂等多种剂型，用于抑制类风湿关节炎所引起的病理变化，防止骨关节的破坏。

Description

血筒素在制备抗类风湿关节炎药物中的应用
相关申请的交叉引用
本申请要求2020年06月12日提交的中国专利申请202010535796.6的权益，该申请的内容通过引用被合并于本文。
技术领域
本发明涉及植物药，具体涉及血筒素在制备抗类风湿关节炎药物中的应用。
背景技术
血筒是一种土家族常用的药用植物,为五味子科南五味子属植物异型南五味子Kadsura heteroclita(Roxb)Craib，常用其藤茎入药。血筒性味甘，微辛，温。具有补血活血、祛风除湿、行气止痛之功效,土家民族民间常用于治疗风湿痹痛、胃脘痛、经痛、骨痛、风湿性关节炎、腰肌劳损、感冒、产后风瘫等症。2005版中国药典收录为“异型南五味子藤茎”，为中华跌打丸的主要成分之一，用于治疗跌打损伤。
血筒素是从血筒中提取的三萜类化合物五内酯E(schisanlactoneE)，现有的研究认为，其具有抗肿瘤作用。
类风湿关节炎(rheumatoidarthritis,RA)是一种病因未明的慢性、以滑膜炎性增生为主的系统性疾病。其特征是手、足小关节的多关节、对称性、侵袭性关节炎症，经常伴有关节外器官受累及血清CRPL、类风湿因子阳性，可以导致关节畸形及功能丧失，导致患者丧失劳动力或者致残。RA的病理特征主要包括滑膜衬里细胞增生、间质大量炎性细胞浸润，以及微血管的新生、血管翳的形成及软骨和骨组织的破坏等。
类风湿关节炎目前尚无法治愈，当前治疗的主要目的在于减轻关节炎症反应，抑制组织病理学改变及骨质破坏，尽可能保护关节和肌肉的功能，最终达到病情完全缓解或降低疾病活动度的目标。目前治疗RA常用的药物是吲哚美辛等非甾体类抗炎药和甲氨蝶呤等抗肿瘤药，但这些药物都有较大的副作用，长期使用会产生较强的不良反应。
发明内容
本发明所要解决的技术问题是提供一种治疗RA的植物药，为RA患者提供一种可以长期使用的植物药成分，丰富RA的治疗手段。
为了实现上述目的，本发明提供了血筒素在制备抗类风湿关节炎药物中的应用。
所述血筒素具有如下式(I)所示的结构:
Figure PCTCN2021099072-appb-000001
本发明还提供了一种抗类风湿关节炎药物，所述抗类风湿关节炎药物以血筒素为主要活性成分。
所述抗类风湿关节炎药物中还含有药学上可接受的盐、酯、糖或赋形剂。
所述抗类风湿关节炎药物可以为片剂、胶囊或针剂。
与现有技术相比，血筒素具有明显的抑制类风湿关节炎成纤维样滑膜细胞活力的作用，抑制类风湿关节炎成纤维样滑膜细胞产生的炎性因子，并能够抑制巨噬细胞产生炎性因子，抑制炎性细胞的浸润与滑膜增生，改善类风湿关节炎的症状，并且使用安全。血筒素提供了一种具有显著的抗类风湿关节炎作用的植物原料药，可以在抗类风湿关节炎药物中使用，并可以制成多种药物剂型。
附图说明
图1是SE与常用抗RA药物对RAFLS细胞活力的影响对比；
图2是SE与常用抗RA药物对RAFLS细胞活力的IC 50值影响对比；
图3是SE与常用抗RA药物对RAFLS产生的TNF-α影响对比；
图4是SE与常用抗RA药物对RAFLS产生的IL-6影响对比；
图5是SE与常用抗RA药物对RAFLS产生的IL-1β影响对比；
图6是SE与常用抗RA药物对RAFLS产生的IL-10影响对比；
图7是SE与常用抗RA药物对LPS诱导小鼠RAW264.7巨噬细胞产生的TNF-α影响对比；
图8是SE与常用抗RA药物对LPS诱导小鼠RAW264.7巨噬细胞产生的IL-6影响对比；
图9是SE与常用抗RA药物对LPS诱导小鼠RAW264.7巨噬细胞产生的IL-1β影响对比；
图10是SE与常用抗RA药物对LPS诱导小鼠RAW264.7巨噬细胞产生的IL-10影响对比；
图11是不同浓度的SE全血溶血率对比；
图12是SE与常用抗RA药物对AIA大鼠脏器组织的影响对比；
图13是SE与常用抗RA药物对AIA大鼠体重影响对比；
图14是SE与常用抗RA药物对AIA大鼠足趾肿胀的影响对比；
图15是SE与常用抗RA药物对AIA大鼠足趾肿胀程度对比；
图16是SE与常用抗RA药物对AIA大鼠的关节炎指数影响对比；
图17是不同剂量的SE对AIA大鼠足趾病理组织影响对比；
图18是不同剂量的SE对AIA大鼠足趾病理评分影响对比；
图19是不同剂量的SE对AIA大鼠足趾骨骼影响CT影像对比；
图20是不同剂量的SE对AIA大鼠血清中的TNF-α影响对比；
图21是不同剂量的SE对AIA大鼠血清中的IL-6影响对比；
图22是不同剂量的SE对AIA大鼠血清中的IL-1β影响对比；
图23是不同剂量的SE对AIA大鼠血清中的IL-17影响对比；
图24是不同剂量的SE对AIA大鼠血清中的IL-23影响对比；
图25是不同剂量的SE对AIA大鼠血液中的ALT影响对比；
图26是不同剂量的SE对AIA大鼠血液中的AST影响对比；
图27是不同剂量的SE对AIA大鼠血液中的ALP影响对比；
图28是不同剂量的SE对AIA大鼠血液中的CREA影响对比；
图29是不同剂量的SE对AIA大鼠血液的WBC影响对比；
图30是不同剂量的SE对AIA大鼠血液的CPR影响对比；
图31是不同剂量的SE对AIA大鼠血液的MONO影响对比；
图32是不同剂量的SE对AIA大鼠血液的RBC影响对比；
图33是不同剂量的SE对AIA大鼠血液的MCV影响对比；
图34是不同剂量的SE对AIA大鼠血液的HCT影响对比；
图35是不同剂量的SE对AIA大鼠血液的PLT影响对比；
图36是不同剂量的SE对AIA大鼠血液的HGB影响对比；
图37是不同剂量的SE对AIA大鼠血液中的WBC影响对比。
具体实施方式
在本文中所披露的范围的端点和任何值都不限于该精确的范围或值，这些范围或值应当理解为包含接近这些范围或值的值。对于数值范围来说，各个范围的端点值之间、各个范围的端点值和单独的点值之间，以及单独的点值之间可以彼此组合而得到一个或多个新的数值范围，这些数值范围应被视为在本文中具体公开。
以下通过实施例对本发明进行详细说明，应当理解的是，此处所描述的具体实施方式仅用于说明和解释本发明，本发明的保护范围并不局限于下述的具体实施方式。
在本发明的具体实施方式中：
所使用的实验细胞和动物：RAFLS细胞购自北京北纳创联生物技术研究院；小鼠RAW264.7巨噬细胞购自中南大学湘雅细胞库；SPF级SD大鼠购自湖南省斯莱克景达实验动物有限公司(合格证号：43004700063752)。
所使用的药物和试剂：高糖DMEM培养基购于美国Gibco公司；1×PBS购于美国Gibco公司；双抗Penicillin-Streptomycin购于美国Gibco公司；胰酶细胞消化液(含0.25％胰酶(Trypsin)和0.02％EDTA,不含Ca2+和Mg2+)购于美国Gibco公司；胎牛血清购于美国Gibco公司；MTT细胞增殖及细胞毒性检测试剂盒购于上海碧云天生物技术有限公司；吲哚美辛(含量≥99.0％)购于西格玛奥德里奇(Sigma-Aldrich，上海)贸易有限公司；TNF-α、IL-6、IL-1β ELISA试剂盒购自武汉博士德生物技术有限公司；热灭活结核分枝杆菌H37Ra(美国Sigma Aldrich公司，20150411)；矿物油(美国Sigma Aldrich公司，M8410)；大鼠TNF-α、IL-1β、IL-6、IL-17、IL-23Elisa试剂盒(武汉博士德生物技术有限公司，201908)；异氟烷气体麻醉剂(上海玉研科学仪器有限公司，S10010533)；苏木精-伊红染色液(北京索莱宝科技有限公司，20190621)；BCA蛋白含量测定试剂盒(宁波有成生物医药科技有限公司，批号：K16G13)；Super ECL化学发光检测试剂盒(宁波有成生物医药科技有限公司，批号：K16G05)；SDS-PAGE凝胶配制试剂盒(宁波有成生物医药科技有限公司，批号：K16G10)；1×TBST(北京索莱宝科学技术有限公司，批号：20190712)；10×TBS(北京索莱宝科学技术有限公司，批号：20190712)；电泳缓冲液(北京索莱宝科学技术有限公司，批号：20190610)；脱脂奶粉(BBI生命科学有限公司，批号：F304BA0037)。其他药物和试剂均为常见的市售产品。
所使用的器械和仪器，德国美耐特游标卡尺(上海美耐特实业有限公司，型号MNT-150)；高分辨率小动物微型CT(美国PerkinElmer-Caliper LS Quantum FX Demo)；高速冷冻离心机(德国Eppendorf 5810R)；组织样本研磨机(德国IKA A11)；匀浆机(德国IKA T10basic)；气体麻醉机(美国赛极威小动物气体麻醉机SurgiVetCDS9000)；电子天平(日本岛津ATY224)；电热鼓风干燥箱(天津泰斯特仪器有限公司，型号：101-1AB)；制冰机(SCOTSMAN，型号：AF100)；核酸蛋白表达成像系统(Bio-Rad Chemi Doc XRS+)。其他均为常见的市售产品。
实施例1
血筒素的提取：
取血筒(异型南五味子)20kg干燥藤茎，阴干后粉碎，用95％乙醇回流提取3次，每次2小时，将滤液合并，减压回收溶剂得到醇提物流浸膏475g，取385g血筒醇提物加入4000mL水使其溶解，依次用石油醚，氯仿，乙酸乙酯，正丁醇萃取，其中得到氯仿层萃取物145g。取氯仿层萃取物145g，硅胶拌样，硅胶湿法装柱(80-100目硅胶3.4kg，10×120cm层析柱)，干法上样，以石油醚-乙酸乙酯梯度洗脱(5:1到0:1)，得到8个流份(流份1-8)。其中流份4重结晶后得到血筒素(Schisanlactone E、SE)1200.0mg。
实施例2
血筒素对类风湿关节炎成纤维样滑膜细胞(RAFLS)的增殖情况的影响：
1、RAFLS细胞培养：RAFLS细胞采用含100U/ml的青霉素、100μg/ml的链霉素作为双抗、10％胎牛血清(FBS)的DMEM/F-12培养基共7mL，放置在6cm细胞培养皿中，于37℃、5％CO 2的培养箱中培养，待细胞贴壁，生长状态良好后，2-3天传代一次。
2、比较SE、甲氨蝶呤、吲哚美辛、青藤碱对RAFLS细胞活力的影响：
取6cm细胞培养皿内对数生长期的RAFLS细胞，用1×PBS清洗2次，取500μL0.25％的胰酶细胞消化液放置于37℃培养箱中消化4min，再用1mL 10％FBS的DMEM培养基终止消化，收集细胞，置于1.5mL EP管中，900rpm离心5min，移除上清，再加入1mL10％FBS的DMEM/F-12培养基，制备单细胞悬浮液，再用10mL含10％FBS的DMEM/F-12培养基制成1×105个/mL单细胞悬液，以每孔100μL均匀接种于96孔板中，置于37℃，5％CO 2培养箱培养。待细胞贴壁良好后，移除培养基，用1×PBS清洗1次，各孔中加入100μM的1％FBS的DMEM/F-12培养基，除空白对照组外，其余各组分别加入终浓度为20μM的甲氨蝶呤(Methotrexate)、吲哚美辛(Indomethacin，简称Indo)、青藤碱(Sinomenine)和SE，作为给药组，每组6个复孔。置于37℃，5％CO 2培养箱中培养48h后，弃去培养基，观察细胞生长状态，随后用含10％MTT的无血清DMEM孵育4h后，弃上清液，每孔加入二甲基亚砜(DMSO)100μL，使用MTT法在酶标仪上于492nm处检测OD值，比较SE和几种常用的抗RA药物在同一浓度下对RAFLS细胞的抑制作用。通过公式：给药组的细胞活力＝(给药组的OD值/空白对照组的OD值)×100％计算给药组的RAFLS细胞活力。
结果如图1所示，与空白对照组(Control组)相比，同一浓度(20μM)的甲氨蝶呤、吲哚美辛及SE均能显著抑制RAFLS细胞的活力，其抑制率分别为：91.28±5.73％，90.25±5.73％，83.62±3.04％。而20μM的青藤碱对RAFLS细胞活力的影响较小，抑制率仅为17.44±2.35％，抑制细胞活力作用弱。
3、比较SE与甲氨蝶呤、吲哚美辛、青藤碱对RAFLS细胞的IC 50值：
取6cm细胞培养皿内对数生长期的RAFLS细胞，用1×PBS清洗2次，取500μL0.25％的胰酶消化液放置于37℃培养箱中消化4min，再用1mL 10％FBS的DMEM/F-12培养基终止消化，收集细胞，置于1.5mL EP管中，900rpm离心5min，移除上清，再加入1mL10％FBS的DMEM/F-12培养基，制备单细胞悬浮液，再用10mL含10％FBS的DMEM培养基制成1×10 5个/mL单细胞悬液，以每孔100μL均匀接种于96孔板中，置于37℃，5％CO2培养箱培养。待细胞贴壁良好后，移除培养基，用1×PBS清洗1次，各孔加入100μM的1％FBS的DMEM/F-12培养基，除空白对照组外，其余各组分别加入甲氨蝶呤、吲哚美辛、青藤碱和SE，使其终浓度分别为0、2.5、5、7.5、10、15、20μM，作为给药组，每种药物每个浓度均设6个复孔。于37℃，5％CO 2培养箱中培养48h后，弃去培养基，观察细胞生长状态，随后用含10％MTT的无血清培养基孵育4h后，弃上清液，每孔加入DMSO100μL，在酶标仪上于492nm处检测OD值。根据OD值与空白组作比较，并计算出药物浓度与细胞活力的标准曲线，计算出IC 50值。细胞活力的计算公式为：给药组的细胞活力＝(给药组的OD值/空白对照组的OD值)×100％。
不同浓度的SE、甲氨蝶呤、吲哚美辛和青藤碱对RAFLSs细胞活力的抑制作用如图2所示，不同浓度的SE对RAFLSs细胞活力的抑制率及所计算出的SE、甲氨蝶呤、吲哚美辛和青藤碱对RAFLSs细胞活力的IC 50值如表1所示。可见，SE、甲氨蝶呤、吲哚美辛、青藤碱对RAFLS细胞活力的IC 50值分别为9.12、2.96、3.81、62.87μM。
表1.SE与常用抗RA药物甲氨蝶呤、吲哚美辛、青藤碱对RAFLSs细胞活力的IC 50值(
Figure PCTCN2021099072-appb-000002
Figure PCTCN2021099072-appb-000003
n＝6)
Figure PCTCN2021099072-appb-000004
Figure PCTCN2021099072-appb-000005
由以上结果可以看出，SE可有效抑制RAFLS的增殖与生长，因而能够在类风湿关节炎发生过程中可抑制滑膜增生。
实施例3
血筒素对炎性因子产生的抑制作用：
1、RAFLS细胞培养：RAFLS细胞采用含100U/ml的青霉素、100μg/ml的链霉素作为双抗、10％胎牛血清(FBS)的DMEM/F-12培养基共7mL，放置在6cm细胞培养皿中，于37℃、5％CO 2的培养箱中培养，待细胞贴壁，生长状态良好后，2-3天传代一次。
2、用Elisa法检测SE对LPS诱导RAFLS产生的炎性因子的影响：
取6cm细胞培养皿对数生长期的RAFLS细胞，用1×PBS清洗2次，取500μL 0.25％的胰酶消化液放置于37℃培养箱中消化4min，再用1mL10％FBS的DMEM培养基终止消化，收集细胞，置于1.5mL EP管中，900rpm离心5min，移除上清，再加入1mL10％FBS的DMEM/F-12培养基，制备单细胞悬浮液，再用10mL含10％FBS的DMEM培养基制成1×10 5个/mL单细胞悬液，以每孔100μL均匀接种于96孔板中，置37℃，5％CO 2培养箱培养。待细胞贴壁良好后，移除培养基，用1×PBS清洗一次，各孔中加入100μM的1％FBS的DMEM培养基，除空白对照组(Model组和Normal组)外，阳性药组(Indo组)加入终浓度为5.0μM的吲哚美辛(Indo)，其余各孔分别加入终浓度为4.5、9.0、18.0μM的SE，分别作为SEL组、SEM组和SEH组，每组6个复孔。于37℃，5％CO 2培养箱培养48h后，除正常组(Normal组)外其余各组均加入LPS使其终浓度为100ng/mL，于37℃，5％CO 2培养箱诱导4h后，收集细胞上清液，3000rpm离心10min，小心取上清液根据TNF-α、IL-6、IL-1β ELISA试剂盒说明书进行检测，在酶标仪上于450nm处检测OD值，根据标准曲线计算出炎性因子的浓度。
如表2、图3-图6所示，与Normal组对比，Model组由LPS诱导的RAFLS上清液中炎性因子TNF-α、IL-6、IL-1β的表达水平显著升高(*P<0.05或**P<0.01)，而IL-10的表达水平显著降低(*P<0.05或**P<0.01)。说明RAFLS本身有炎性因子产生，而LPS诱导4h后能增加RAFLS细胞中炎性因子的分泌。与Model组相比，吲哚美辛在5.0μM的浓度下能显著降低RAFLS中炎性因子TNF-α、IL-6、IL-1β的表达水平(*P<0.05或**P<0.01)，并显著升高IL-10的表达水平(*P<0.05或**P<0.01)。与Model组相比，SE在4.5、9.0、18.0μM浓度下刺激RAFLS细胞24h后，均能显著抑制其分泌的炎性因子TNF-α、IL-6、IL-1β的表达(*P<0.05或**P<0.01)，且升高IL-10的表达水平(*P<0.05或**P<0.01)。
表2 SE对LPS诱导的RAFLS上清液中TNF-α、IL-1β、IL-6抑制率的影响(
Figure PCTCN2021099072-appb-000006
n＝6)
Figure PCTCN2021099072-appb-000007
Figure PCTCN2021099072-appb-000008
3、小鼠RAW264.7巨噬细胞的培养：
小鼠RAW264.7巨噬细胞采用含100U/ml的青霉素、100μg/mL的链霉素作为双抗、10％胎牛血清(FBS)的高糖DMEM培养基共7mL，置于6cm细胞培养皿中，于37℃、5％CO 2的培养箱中培养，待细胞贴壁，生长状态良好后，2-3天传代一次。
4、用Elisa法检测SE对LPS诱导小鼠RAW264.7巨噬细胞产生的炎性因子的影响：
取对数生长期的RAW264.7细胞，用1×PBS清洗2次，取500μL 0.25％的胰酶细胞消化液放置于37℃培养箱中消化4min，再用1mL10％FBS的DMEM培养基终止消化，收集细胞，置于1.5mL EP管中，900rpm离心5min，移除上清，再加入1mL10％FBS的DMEM培养基，制备单细胞悬浮液，再用10mL含10％FBS的DMEM培养基制成1×10 5个/mL单细胞悬液，以每孔100μL均匀接种于96孔板中，置37℃，5％CO 2培养箱培养。待细胞贴壁良好后，移除培养基，用1×PBS清洗一次，各孔中加入100μM的1％FBS的DMEM培养基，除空白对照组(Model组和Normal组)外，阳性药组(Indo组)加入终浓度为5.0μM的吲哚美辛，其余各孔再加入终浓度分别为4.5、9.0、18.0μM的SE，分别作为SEL组、SEM组和SEH组，每组6个复孔。于37℃，5％CO 2培养箱培养48h后，除正常组(Normal组)外其余各组均加入LPS使其终浓度为100ng/mL，于37℃，5％CO 2培养箱诱导4h后，收集细胞上清液3000rpm离心10min，小心取得上清液，根据TNF-α、IL-6、IL-1β ELISA试剂盒说明书进行检测，在酶标仪上于450nm处检测OD值，根据标准曲线计算出炎性因子的浓度。
实验结果如图7-图10所示，与Normal组对比，Model组由LPS诱导的RAW264.7上清液中炎性因子TNF-α、IL-6、IL-1β的表达水平显著升高(*P<0.05或**P<0.01)，而IL-10的表达水平显著降低(*P<0.05或**P<0.01)。说明RAFLS本身有炎性因子产生，而LPS诱导4h后能增加RAFLS细胞中炎性因子的分泌。与Model组相比，吲哚美辛在5μM的浓度下能显著降低RAFLS中炎性因子TNF-α、IL-6、IL-1β的表达水平(*P<0.05或**P<0.01)，并显著升高IL-10的表达水平(*P<0.05或**P<0.01)。与Model组相比，SE在4.5、9.0、18.0μM浓度下刺激RAFLS细胞24h后，均能显著抑制其分泌的炎性因子TNF-α、IL-6、IL-1β的表达(*P<0.05或**P<0.01)，且升高IL-10的表达水平(*P<0.05或**P<0.01)。
由以上结果可以看出，SE可有效抑制RAFLS和小鼠RAW264.7巨噬细胞产生的炎性因子，并提升其IL-10的表达水平，因而能够在类风湿关节炎发生过程中可抑制炎性反应的产生，减轻类风湿关节炎的症状和对机体的损伤。
实施例4
血筒素对佐剂型关节炎(AIA)大鼠模型的影响：
1、血筒素溶血性实验：
将大鼠全血样在4℃下以3000rpm离心5min，并用PBS洗涤5次以收集纯红细胞。将50μL4％红细胞(v/v)与950μL不同浓度(0.21、0.42、0.84、1.68和3.36mg/mL)的SE(分散剂均为pH＝7.4的PBS)，以及50μL4％红细胞(v/v)与950μL的PBS相混合，所得混合液在37℃下孵育4h。阳性对照是利用与纯水混合的红细胞作为100％溶血。将混合液在4℃下以3000rpm离心5min，使用UV-Vis分光光度计测定540nm处上清液的吸光度。用公式：Hemolysis(％)＝(I/I 0)×100％计算溶血百分比。其中，Hemolysis表示溶血率，I表示由不同浓度的SE加入红细胞悬浮液后的上清液吸光度，I 0表示在纯水中完全溶血的吸光度。所有溶血实验重复三次进行。
如图11所示，大鼠红细胞的纯水中全部溶血，溶血率为100％，在1×PBS中不发生溶血现象，溶血率为0％。红细胞在SE浓度0.21、0.42、0.84、1.68、3.36mg/mL的条件下溶血率分别为1.04、1.05、1.06、1.41、1.45％。可见SE符合ISO规定的溶血率＜5％ 的标准，符合作为药品使用的该项安全性标准。
2、AIA大鼠模型的建立、药物干预及检测样本的提取：
取SPF级SD大鼠60只，体重在70-90g之间，饲养于IVC屏障系统内，恒温恒湿，适应性喂养一周后。将大鼠随机分成6组(每组6只大鼠)，分别为：空白组(Normal)、模型组(Model)、吲哚美辛组(Indo 1.0mg/kg)、SE低剂量组(SE 0.21mg/kg)、SE中剂量组(SE 0.42mg/kg)、SE高剂量组(SE 0.84mg/kg)。
SD大鼠适应性喂养并分组后第7天，除空白组外，模型组及其余各药物组大鼠尾根部皮下注射0.1mL含200μg热灭活结核杆菌(Mtb)的完全弗氏佐剂(CFA)建立AIA模型，造模后，各组分别尾静脉注射生理盐水1mL(模型组)，吲哚美辛(1mL，1mg/kg)，SE低剂量(1mL，0.21mg/kg)，SE中剂量(1mL，0.42mg/kg)，SE高剂量(1mL，0.84mg/kg)。每间隔1天治疗1次，直至造模后第30天结束。自造模后第1天起，每天观察各组大鼠关节炎发病情况，每3天称量大鼠体重，用游标卡尺测量大鼠后足肿胀厚度并进行关节炎指数评分(分别对大鼠的四个足趾肿胀情况予以评分，评分标准：0分：无关节肿胀；1分：小趾关节稍红肿；2分：趾关节和足趾关节红热肿胀；3分：踝关节以下全部红热肿胀；4分：包括踝关节以内全部红热肿胀。四个足趾的得分相加得到关节炎指数评分)。造模后第18天，各组大鼠通过眼眶采血，4℃、3500rpm条件下离心15min，分离上层血清，保存于-80℃冰箱备用。造模后的第21天，将大鼠用小动物气体麻醉机进行麻醉，用小动物CT扫描各组大鼠后足关节，评价关节肿胀及骨破坏程度。造模后第31天，麻醉大鼠，腹主动脉取血法收集大鼠血液，4℃、3500rpm条件下离心15min，将获得的大鼠全血取一部分，用全自动血常规检测仪分析各组每只大鼠血液的血常规指标，另一部分血液分离上层血清用于ALT、AST及炎性因子等血液生化指标测定。对大鼠实施安乐死并收集主要器官(心脏、肝脏、脾脏、肺和肾脏)，并从踝关节以上截取足趾，各取一半用4％多聚甲醛固定，另一部分放置于-80℃冰箱冻存以便用于提取蛋白。
从光学显微镜10×40倍镜下大鼠器官切片，如图12所示，与吲哚美辛组和空白组相比，SE各个剂量组的心脏、肝脏、脾脏、肺、肾脏组织均无明显变化，说明SE对AIA模型大鼠的各个脏器无损害作用。
如图13所示，与空白组大鼠相比，从AIA造模后的第9天起，模型组大鼠食欲减退，后足开始肿胀严重，活动不便，活动量减少，灵活性变差等关节炎症状，关节出现畸形，体重增长变缓慢，且在关节炎严重时期，即第9-15天大鼠体重增长停滞并有呈下降趋势。吲哚美辛组给予吲哚美辛1.0mg/kg治疗后，大鼠的生活质量显著得以改善。SE各个剂量组在给予SE 0.21、0.42、0.84mg/kg的剂量治疗后，大鼠体重保持增长趋势，在高剂量(0.84mg/kg)组，大鼠体重的增长趋势与吲哚美辛组的大鼠体重接近。可见，SE可显著降低AIA模型大鼠关节炎症对大鼠生活的影响，改善大鼠的生存与生活质量，高剂量SE的治疗效果与吲哚美辛相近。
如图14-图16所示，造模后第9天起，与空白组相比，模型组大鼠足趾肿胀开始发生，大鼠前、后足趾均逐渐出现红肿热痛等炎症症状。造模后第21天，模型组每只大鼠前后足趾均肿胀明显，且达到了最大肿胀程度，大鼠四肢关节出现畸形，明显灵活度变差，活动量、进食量均显著减少。与模型组相比，给予SE治疗的大鼠足趾的肿胀程度呈剂量依赖性地减小。SE各剂量组均能显著减少关节炎的发生率，其中模型组发病率为100％且发病最为严重，SE 0.42mg/kg剂量组大鼠发病率为75％，SE 0.84mg/kg剂量组为20％，发病程度呈剂量依赖性地减轻。从测得的大鼠足趾肿胀厚度看，吲哚美辛组经吲哚美辛治疗后的大鼠足趾肿胀不明显，SE在0.21、0.42、0.84mg/kg剂量下能显著降低AIA模型大鼠足趾肿胀，降低关节炎指数，并呈一定的剂量依赖性，差异具有统计学意义(*P<0.05,**P<0.01或***P<0.001)。
将经福尔马林固定的大鼠后足趾进行脱钙处理、石蜡包埋，并切片后用苏木精-伊红(HE)染色，在光学显微镜10×40倍镜下观察，并根据关节腔变窄程度、滑膜组织增生程度、 炎性细胞浸润程度和血管翳的增生程度进行病理评分。评分方法为：关节腔隙正常、无明显滑膜组织增生、无炎性细胞浸润、无血管翳新生病理均得0分；关节腔隙稍狭窄、轻微的滑膜组织增生、少许的炎性细胞浸润、极轻微的血管翳新生均得1分；关节腔隙狭窄、中度的滑膜组织增生、大量的炎性细胞浸润、轻微的血管翳新生均得2分；关节腔隙明显狭窄、严重的滑膜组织增生、大量的炎性细胞浸润、严重的血管翳新生均得3分。将关节腔变窄程度、滑膜组织增生程度、炎性细胞浸润程度和血管翳的增生程度的得分相加，得到大鼠后足趾的病理评分。如图17、图18所示，空白组大鼠关节细胞结构完整，排列整齐，关节软骨完整光滑、软骨下骨组织结构清晰；与空白组比较，模型组大鼠关节肿胀明显，大量炎性细胞浸润，滑膜下血管和侵蚀性血管翳形成严重的结缔组织增生，关节间隙显著狭窄，局部软骨细胞脱落，软骨和骨破坏严重(P<0.001)；与模型组相比，SE 0.21mg/kg剂量组，滑膜中等程度增生，关节腔较狭窄，可见炎性细胞浸润，关节腔软骨细胞肿胀。吲哚美辛组与SE 0.42、0.84mg/kg剂量组大鼠足趾组织滑膜细胞轻度增生，可见个别炎性细胞于组织间，关节腔隙未发生明显变化(*P<0.05或**P<0.01)。说明SE各剂量均能有效地保护AIA模型大鼠踝关节，抑制炎性细胞的浸润与滑膜增生，阻碍关节腔变狭窄及血管翳的生成。
造模后的第21天的CT影像如图19所示，可见空白组大鼠趾间关节、跖趾关节对应关系良好，关节间隙清晰，未见狭窄或增宽，关节面光滑。与空白组比较，模型组大鼠后足趾各个关节出现严重骨侵蚀，大量骨赘增生，关节腔隙狭窄甚至消失，关节严重畸形。与模型组相比，吲哚美辛组及SE中、高剂量组大鼠后足趾关节骨侵蚀、关节腔隙狭窄程度都较轻，关节结构较完整，提示SE对大鼠足趾关节具有较好的保护作用。
将大鼠全血离心后获得的血清，用Elisa试剂盒检测血清当中TNF-α、IL-6、IL-1β、IL-17、IL-23等炎性因子的表达水平。检测结果如图20-图24所示，与空白组相比，模型组大鼠血清中TNF-α、IL-6、IL-1β、IL-17、IL-23等炎性因子水平明显升高( #P<0.05)，与模型组比较，在中、高剂量(0.42mg/kg及0.84mg/kg)组均能显著下调TNF-α、IL-6、IL-1β、IL-17、IL-23炎性因子水平(*P<0.05或**P<0.01)，SE低剂量(0.21mg/kg)能显著降低TNF-α、IL-6、IL-17、IL-23的表达水平(*P<0.05或**P<0.01)。作为阳性对的照吲哚美辛组能显著抑制TNF-α、IL-6、IL-1β、IL-17、IL-23的表达(*P<0.05或**P<0.01)。提示SE可能通过抑制关键炎性细胞因子表达而发挥抗AIA药效作用。
对大鼠血清的血生化检测结果如图25-图28所示，与空白组相比，模型组大鼠血液中ALT、AST、ALP、CREA均显著升高，说明，佐剂对关节炎大鼠的肝肾功能有一定的损害( #P<0.05)。与模型组大鼠相比，SE各剂量组(0.21、0.42、0.84mg/kg)均能显著降低AIA模型大鼠血液中ALT、AST、ALP、CREA的含量(*P<0.05或**P<0.01)。说明SE在治疗关节炎的过程中保护了大鼠的肝脏、肾脏组织。
对大鼠全血的进行血常规检测。血常规对血细胞分析的结果如图29-图31所示，与正常组相比，模型组大鼠WBC、C型反应性蛋白(C-reactive protein level,CRPL)、单核细胞(Monocyte，MONO)均显著升高，说明AIA模型大鼠血液中炎症反应严重，炎性细胞增多，机体自身抵抗炎症作用力强( #P<0.05)。与模型组对比，阳性药吲哚美辛治疗组的大鼠血液中炎性细胞比例显著减少。经过治疗后，在0.21、0.42、0.84mg/kg的SE剂量条件下治疗后的大鼠炎性细胞比例显著下降(*P<0.05或**P<0.01)，说明SE具有较强的抗炎作用。从两次血样结果可见，在关节炎大鼠足爪肿胀最大时期(第21d)，体内炎症反应最严重，随着关节炎的逐渐恢复，大鼠体内炎症反应减轻(第31d)。如图32-图34所示，与正常组相比，模型组大鼠全血中RBC、MCV、HCT比例显著下降( #P<0.05)，造血功能出现异常而导致大鼠出现贫血现象，具体表现为大鼠毛色枯黄、活动迟钝、进食少。据文献报道，罹患关节炎患者有贫血现象，我们的结果支持了这一结论。与模型组相比，SE在0.21、0.42、0.84mg/kg剂量条件下治疗后的大鼠血液中RBC、MCV、HCT比例显著升高(*P<0.05或**P<0.01)。说明SE能改善大鼠造血功能，治疗关节炎过程中恢复大 鼠生理机能。如图35-图37所示，与正常组相比，AIA模型组大鼠全血中PLT、MPV显著升高，血红蛋白(Hemoglobin,HGB)降低(P<0.05)，取血时同样发现模型组大鼠血液黏度高，易凝固，因此，AIA造模后，会使大鼠血小板比例升高，造成血液容易凝固，导致血液淤积。与模型组大鼠相比，SE在0.21、0.42、0.84mg/kg剂量条件下治疗后的大鼠PLT、MPV比例显著降低，HGB升高(*P<0.05或**P<0.01)，可改善AIA模型大鼠血瘀症状，促进血液流通，降低血液黏度。综上所述，血筒素(SE)能有效的抑制类风湿关节炎成纤维样滑膜细胞的增生，并有效抑制脂多糖(LPS)诱导的RAFLS的炎性因子TNF-α、IL-1β、IL-6的产生，并升高IL-10的表达水平。还可以有效抑制LPS诱导的RAW264.7中的炎性因子TNF-α、IL-1β、IL-6的产生，并升高IL-10的表达水平，显示出其抗炎作用。在与目前公认有效并广泛使用的类风湿关节炎治疗药吲哚美辛对比进行的动物实验中可以看出，SE低、中、高剂量(0.21、0.42、0.84mg/kg)在比吲哚美辛(剂量为：1.0mg/kg)更低的剂量条件下能有效的抑制AIA诱导的大鼠关节炎的发生率，降低大鼠足趾肿胀度，通过病理切片对大鼠足趾进行组织病理学观察，发现SE各剂量条件下均有效抑制炎性细胞的浸润与滑膜增生，阻碍关节腔变狭窄及血管翳的生成。通过小动物CT扫描对大鼠足趾关节进行放射学观察，发现SE各剂量条件下均明显改善大鼠后足趾关节骨及软骨侵蚀、减轻关节腔隙狭窄程度，保护关节结构趋向完整，提示SE对大鼠足趾关节具有较好的保护作用。结果显示，SE可以作为治疗类风湿关节炎的药物来使用，为类风湿关节炎的治疗提供了一种全新的、疗效明确的、使用安全的植物来源药物。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。

Claims (4)

  1. 血筒素在制备抗类风湿关节炎药物中的应用。
  2. 一种抗类风湿关节炎药物，其特征在于，以血筒素为主要活性成分。
  3. 根据权利要求2所述的抗类风湿关节炎药物，其特征在于，还含有药学上可接受的盐、酯、糖或赋形剂。
  4. 根据权利要求2所述的抗类风湿关节炎药物，其特征在于，所述的药物为片剂、胶囊或针剂。
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