WO2018145268A1 - 降压药在预防和治疗骨坏死中的应用 - Google Patents

降压药在预防和治疗骨坏死中的应用 Download PDF

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WO2018145268A1
WO2018145268A1 PCT/CN2017/073118 CN2017073118W WO2018145268A1 WO 2018145268 A1 WO2018145268 A1 WO 2018145268A1 CN 2017073118 W CN2017073118 W CN 2017073118W WO 2018145268 A1 WO2018145268 A1 WO 2018145268A1
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osteonecrosis
yap
hif
angiotensin
glucocorticoid
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PCT/CN2017/073118
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French (fr)
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张长青
朱弘一
陶诗聪
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上海市第六人民医院
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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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/401—Proline; Derivatives thereof, e.g. captopril
    • 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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • 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
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease

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  • the present application relates to antihypertensive drugs, such as angiotensin converting enzyme (ACE) inhibitors or angiotensin II receptor blockers for preventing or treating osteonecrosis; and also relates to HIF-1a agonists for preventing or treating osteonecrosis Application of drugs
  • ACE angiotensin converting enzyme
  • angiotensin II receptor blockers for preventing or treating osteonecrosis
  • HIF-1a agonists for preventing or treating osteonecrosis
  • Bone tissue itself is the most common cause of osteonecrosis. At present, researchers generally believe that the ability of bone tissue regeneration and repair, blood supply disorders are an important mechanism of osteonecrosis. Hormones and alcohol are the most common in vitro factors that cause the above-mentioned pathophysiological processes, and are also the most common cause of osteonecrosis in China.
  • Symptoms and signs of femoral head necrosis are diverse, and the time of onset of the disease varies from one to the other. However, various clinical manifestations are not unique to femoral head necrosis, and many hip joint diseases can occur. In other words, it is difficult to diagnose the femoral head necrosis by subjective symptoms and clinical examination. In addition, early X-ray films were not positively detected. With the progress, trabecular trabeculae occurred in the weight-bearing area, interrupted, and the posterior subchondral capsule of the femoral head and the inclusions were hardened. The X-ray showed a new lunar sign to confirm the diagnosis of femoral head necrosis.
  • osteonecrosis at the time of patient visit has often reached a more serious stage, further increasing the difficulty of treatment. Since there is no effective drug for the treatment of femoral head necrosis, the treatment of femoral head necrosis can only rely on surgery. In the early stage, surgery to preserve the femoral head can be taken, such as: core decompression, vascular bone grafting, etc. In the inevitable case of the late stage, artificial joint replacement is required. However, in general, surgical therapy is not accepted by many patients because of its pain, high cost, long recovery period, and long-term effects.
  • Glucocorticoids are widely used to treat a variety of diseases, including autoimmune diseases, acute lymphoblastic leukemia, kidney syndrome, and severe inflammation.
  • long-term or high-dose administration of glucocorticoids can cause health problems.
  • Osteonecrosis also known as avascular necrosis, is one of several common serious complications that occurs after the glucocorticoid treatment has been impeded by the supply of blood to bone tissue.
  • the incidence of osteonecrosis in patients with systemic lupus erythematosus (1, 2) who received glucocorticoid therapy ranged from 9.5% to 37%.
  • the incidence of osteonecrosis is 4.6-20% (3, 5) of the organ recipient.
  • glucocorticoids inhibit the synthesis of hypoxia-inducible factor 1 ⁇ (HIF-1 ⁇ ) and angiogenic factors in human peripheral lung epithelial cells (6).
  • HIF-1 ⁇ hypoxia-inducible factor 1 ⁇
  • angiogenic factors in human peripheral lung epithelial cells (6).
  • the destruction of vascular regeneration may be related to the pathogenesis of osteonecrosis.
  • HIF-1 ⁇ and angiogenic factors can lead to osteonecrosis in rats.
  • angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers can be used to prevent osteonecrosis in rats administered high doses of glucocorticoids.
  • the main content of this application is the new role of angiotensin converting enzyme (ACE) inhibitor or angiotensin II receptor blocker for preventing or treating osteonecrosis.
  • ACE angiotensin converting enzyme
  • the present application relates to the use of an angiotensin converting enzyme (ACE) inhibitor or an angiotensin II receptor blocker for the preparation of a medicament for preventing or treating osteonecrosis.
  • ACE angiotensin converting enzyme
  • the present application relates to a method of preventing or treating osteonecrosis comprising administering a therapeutically effective amount of an angiotensin converting enzyme (ACE) inhibitor or an angiotensin II receptor blocker to Subjects with this need.
  • ACE angiotensin converting enzyme
  • the osteonecrosis is glucocorticoid-induced osteonecrosis.
  • the above osteonecrosis is caused by YAP-mediated inhibition of HIF-1a.
  • an angiotensin converting enzyme (ACE) inhibitor or an angiotensin II receptor blocker prevents or treats osteonecrosis by inhibiting activation of YAP.
  • ACE angiotensin converting enzyme
  • the angiotensin converting enzyme (ACE) inhibitor is preferably captopril, and the angiotensin II receptor blocker is preferably losartan.
  • the present application relates to the use of a HIF-1a agonist for the manufacture of a medicament for preventing or treating osteonecrosis.
  • the present application relates to a method of preventing or treating osteonecrosis using a HIF-1a agonist.
  • the main difficulty in drug treatment of osteonecrosis is that due to the blood supply disorder in the osteonecrosis area, drugs that promote bone repair often have difficulty reaching the necrotic area. In response to this situation, we have improved the blood supply to the necrotic area as the main direction for the treatment of osteonecrosis.
  • Vascular injury can lead to ischemia and hypoxia.
  • Cells in ischemic and hypoxic tissue up-regulate their own HIF-1a, thereby promoting blood Tube new students.
  • glucocorticoids activate YAP protein to inhibit HIF-1a in the body, thereby inhibiting angiogenesis. This inhibition breaks the dynamic balance of vascular damage and angiogenesis in the body, eventually leading to the disappearance of bone tissue blood supply, which in turn causes osteonecrosis.
  • an angiotensin-converting enzyme (ACE) inhibitor or an angiotensin II receptor blocker to inhibit YAP, thereby increasing angiogenesis and preventing glucocorticoid-induced osteonecrosis.
  • ACE angiotensin-converting enzyme
  • angiotensin II receptor blockers we promote the repair of necrotic areas by angiotensin-converting enzyme (ACE) inhibitors or angiotensin II receptor blockers.
  • Glucocorticoid-induced osteonecrosis is caused by YAP-mediated inhibition of HIF-1a, angiotensin-converting enzyme (ACE) inhibitor or angiotensin II receptor blocker prevents osteonecrosis by inhibiting YAP activation .
  • ACE angiotensin-converting enzyme
  • angiotensin II receptor blocker prevents osteonecrosis by inhibiting YAP activation .
  • the present application relates to the use of a YAP inhibitor for the preparation of a medicament for preventing or treating osteonecrosis.
  • the YPA inhibitor is selected from the group consisting of captopril and losartan.
  • glucocorticoids Long-term or high-dose administration of glucocorticoids can lead to osteonecrosis, which usually results in osteonecrosis of the femoral head, resulting in joint damage and limited movement.
  • This article is the first study of glucocorticoid-induced osteonecrosis caused by inhibition of HIF-1a and some angiogenic factors.
  • the present application identified YAP (the effector of the Hippo pathway) as a downstream mediator of glucocorticoid-induced signal transduction, exhibiting the activity of inhibiting HIF-1a by the expression of GNB2L1.
  • the present application further demonstrates that the cross talk between the glucocorticoid receptor and the Hippo signal contributes to the activation of YAP by glucocorticoids.
  • angiotensin converting enzyme (ACE) inhibitor or an angiotensin II receptor blocker lowering serum cholesterol levels
  • ACE angiotensin converting enzyme
  • an angiotensin II receptor blocker lowering serum cholesterol levels
  • FIG. 1 Glucocorticoids inhibit HIF-1 ⁇ to prevent angiogenic factor secretion.
  • (B) Lysates of rat bone tissue treated with or without methylprednisolone (MPS) were analyzed by immunoblotting with HIF-1 ⁇ and VEGF antibodies (n 7 per group). The statistical results of protein content compared with the control group are shown in the figure.
  • MPS methylprednisolone
  • each group of four columns represents normal oxygen content, low oxygen content, normal oxygen content + DEX, low oxygen content + DEX from left to right.
  • F Levels of angiogenic factor mRNA in BMSCs after stimulation of 1 ⁇ M DEX for 24 hours in normal or hypoxic conditions. *P ⁇ 0.05 vs. normal oxygen content. **P ⁇ 0.01 vs. normal oxygen content. ##P ⁇ 0.01Compared with low oxygen content, student t test.
  • each group of four columns from left to right represents normal oxygen content, low oxygen content, normal oxygen content + DEX, low oxygen content + DEX
  • G receiving BAY 87-2243 (4 mg / kg) and Rats after six months of axitinib (20 mg/kg) developed osteonecrosis.
  • Each set of bone-related parameters included bone volume/tissue volume (BV/TV), number of trabeculae (Tb.N), thickness (Tb.Th), and statistical results of separation (Tb.Sp). **P ⁇ 0.01, Student's t test.
  • the four columns in the histogram represent the control, MPS, BAY 87-2243, and axitinib from left to right.
  • H Typical pathology areas for each group. In the MPS, BAY 87-2243 and axitinib groups, trabecular bone was lost and replaced by adipose tissue.
  • FIG. 1 Glucocorticoid-induced YAP activation.
  • A Microarray data (repeated three times per group) The heat map shows that after 24 hours of DEX treatment, three YAP downstream genes (ANKRD1, CTGF, Cyr61) were up-regulated and VEGF was significantly down-regulated.
  • B Activation of the three downstream genes was determined by qPCR assay. **P ⁇ 0.01 vs. 0 nM, Student's t test. The four columns in each group in the figure represent the expression levels of mRNA at 0nMDEX, 10nMDEX, 100nMDEX and 1000nMDEX from left to right.
  • YAP subcellular localization was monitored by immunofluorescence of BMSCs treated with DEX. Nuclear localization was observed after 3 hours of treatment with 100 nM DEX. Scale bar, 100 ⁇ m.
  • D BMSCs were stimulated with four different concentrations of DEX (0 nM, 10 nM, 100 nM, 1,000 nM) for 3 hours. The YAP dephosphorylation reaction in BMSCs was positively correlated with the concentration of DEX.
  • FIG. 3 Glucocorticoid-induced inhibition of HIF-1 ⁇ signaling pathway is mediated by activated YAP.
  • A Infecting YAP or empty lentivirus BMSCs under normal oxygen or hypoxic conditions. Overexpression of YAP under hypoxic conditions prevents aggregation of HIF-1 ⁇ .
  • B BMSCs infected with viral shYAP or control shRNA were challenged with 1 ⁇ M DEX under hypoxic conditions.
  • C Candidate genes were detected after YAP overexpression or 100 nM DEX treatment of BMSCs. GNB2L1 was mostly upregulated in both experiments. **P ⁇ 0.01. See also Figure S3.
  • (D) Rat bone tissue GNB2L1 Protein levels were analyzed by immunoblotting (n 7 per group). The statistical results of the protein content of the control group are shown. **P ⁇ 0.01 vs. control group, Student's t test.
  • (F) Protein levels of YAP and GNB2L1 in bone tissue of patients with osteonecrosis were significantly increased relative to healthy subjects. Lysates of uninfected bone tissue were analyzed by indicator antibody spotting (n 24 per group). The statistical results of protein content relative to healthy subjects are shown. **P ⁇ 0.01 vs. control group, Student's t test.
  • FIG. 4 Glucocorticoid-induced YAP activation is cross-talked by GR and LATS1/2.
  • A The expression of ANKRD1, CTGF and Cyr61 was up-regulated by GR in BMSCs by siRNA knockdown. **P ⁇ 0.01 vs. siCTL, Student's t test. The leftmost column in each set of columns in the figure represents the siCTL.
  • B S211A GR gene engineered expression blocked YAP nuclear localization when BMSCs were treated with 100 nM DEX. The scale bar is 100 ⁇ m.
  • C Wild-type genetically engineered expression does not block DEX-induced YAP dephosphorylation as with S211A GR.
  • Endogenous MST1 was immunoprecipitated with cell lysate treated with or without DEX (100 nM, 3 hours), and MST1 kinase activity was determined using GTS-MOB1 as a substrate. MST1/2 phosphorylation associated with kinase activity did not change after DEX treatment.
  • DEX induces YAP phosphorylation by activating LATS1. Endogenous LATS1 was immunoprecipitated by treatment of BMSCs as indicated. LATS1 activity was elevated after DEX treatment (100 nM, 3 hours) or GRs knockout. However, blocking DEX-induced S211A GR gene engineered expression increased LATS1 kinase activity.
  • Figure S1 Protein content of YAP, GNB2L1, HIF-1 ⁇ and VEGF in bone tissue of patients and healthy subjects. All patients received glucocorticoid therapy for medical purposes until the sample acquisition date. The protein content was detected by the spotting method.
  • FIG. 1 Protein levels of YAP, GNB2L1, HIF-1 ⁇ and VEGF in bone tissue after MPS intramuscular injection of MPS. After 24 hours of MPS treatment, the protein levels of YAP, GNB2L1, HIF-1 ⁇ and VEGF were evaluated by Western blotting.
  • FIG. 1 mRNA levels of HIF-1A after DEX treatment or YAP overexpression. There was no significant change in HIF-1A expression in BMSCs after DEX treatment (100 nM) or overexpression of YAP.
  • Figure S mRNA levels of candidate genes that inhibit HIF-1 ⁇ . After YAP overexpression or 100nMDEX treatment in BMSCs, mRNA levels of candidate genes were evaluated by qPCR.
  • FIG. 1 Schematic diagram of the mechanism of angiotensin-converting enzyme (ACE) inhibitor or angiotensin II receptor blocker for preventing and treating osteonecrosis
  • ACE angiotensin-converting enzyme
  • FIG. 1 Rats were tested for the concentration of the protein shown in bone tissue one day after administration as shown.
  • the hormone dose was the same as before, ARB was given to losartan at a dose of 1 mg/kg, and ACEI was given captopril at a dose of 1 mg/kg.
  • the mode of administration is gavage.
  • levels of HIF-1 ⁇ and VEGF in bone tissue returned to near normal levels compared to the single hormone group. This indicates that the above two drugs can effectively reverse the angiogenesis inhibition caused by glucocorticoids.
  • captopril Since the mechanism of action of captopril is to reduce the production of angiotensin II in the blood, the content of HIF-1 ⁇ and VEGF is returned to a similar level in the hormone group after intravenous administration of angiotensin II.
  • the above experiments further confirmed that glucocorticoids inhibit angiogenesis in bone tissue through the AGTR1 receptor.
  • Figure S12. shows that both ACEI and ARB drugs prevent the occurrence of hormone-induced femoral head necrosis.
  • Figure S13 shows that both ACEI and ARB drugs can significantly reduce the incidence of femoral head necrosis.
  • ARB represents losartan at a dose of 1 mg/kg and ACEI represents captopril at a dose of 1 mg/kg.
  • the model was the same as before, and the first time the hormone injection was given for 6 weeks, the two preventive drugs were given daily.
  • the use of ACEI and ARB can effectively prevent the occurrence of osteonecrosis.
  • BMSCs were isolated from healthy subjects (age, 30-50 years old) who had undergone amputation due to severe trauma. Briefly, cancellous bone was obtained at the time of surgery and was first rinsed with culture medium and then transferred to a T75 container. BMSCs are separated by adhesion. BMSCs were cultured in ⁇ -MEM (granulation) and supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 ° C and 5% CO 2 . The following chemicals were used in this study: dexamethasone (Sigma-Aldrich). For hypoxia stimulation, cells were cultured in ⁇ -MEM containing 10% FBS, with or without dexamethasone, and cultured for 24 hours at 37 ° C, 5% CO 2 and 1% O 2 .
  • FBS fetal bovine serum
  • Enzyme-linked immunosorbent assay All ELISA kits for this study were obtained from Boster Biological Technology. The ELISA test was performed according to the product guidelines. Samples were added to 96-well plates for 90 minutes at 37 ° C, which were pre-coated with antibodies in the respective kit components. Then, the biotin-binding antibody was added at 37 ° C for 60 minutes and then washed three times with TBS. After the addition of the ABC solution, the plates were washed five times with TBS. Prior to the assay, the TMB solution was added at 37 ° C for less than 30 minutes and then the reaction was terminated with TMB stop buffer.
  • Microarray technology Microarray experiment PrimeView TM Human Gene expression array. Robust multi-array average (RMA) derivation is applied to Bioconductor's 'affy' package for raw intensity data for inter-chip standardization. Then perform a 'limma' package to select differentially expressed genes (DEGs). Selected gene expression values are converted to Z-scores and then aggregated for generating heat maps based on Euclidean distance and average linkage grades.
  • RMA multi-array average
  • RNA integrity and quantity were analyzed by NanoDrop 2000. Removal of contaminant DNA and reverse transcription was performed on EasyScript One Stepg DNA Removal and cDNA Synthesis SuperMix (Beijing TransGen Biotech Co., Ltd). The resulting cDNA was diluted reasonably and used for qPCR reaction using TransStart Tip Green qPCR SuperMix (Beijing TransGen Biotech Co., Ltd.) using ABI 7900HT (Applied) Biosystems) and analyzed with SDS 2.4 (Applied Biosystems). Each experiment was performed at least three times. In this study, GAPDH and ⁇ -actin were used as housekeeping genes for standardization of results. The PCR sequence of the human sample PCR is reported in Appendix Table 2.
  • VEGF antibodies were obtained from Boster Biological Technology.
  • YAP1 mouse antibody and HIF-1 ⁇ antibody were obtained from Wuxi UcallM Biotechnology.
  • Glucocorticoid receptor, YAP rabbit, MST1 rabbit, MST2, LATS1 (C66B5), ⁇ -actin (8H10D10), phospho-MOB1 (Thr12), phospho-MST1 (Thr183)/MST2 (Thr180) and phospho-YAP (Ser127) (D9W2I) antibody was obtained from Cell Signaling Technology.
  • MST1 mouse antibody was cultured in the chamber. All antibodies were diluted based on specific assays according to product guidelines.
  • BMSCs were grown on coverslips at a suitable density and then processed as indicated. After 15 minutes of fixation with 4% paraformaldehyde, BMSCs were permeabilized with 0.1% Triton X-100 for 15 minutes and blocked with 10% FBS for 1 hour in PBS. The initial antibody was then added for 1 hour at 37 ° C, followed by three washes with PBS for five minutes each. The cells were then incubated for 30 minutes and conjugated with a separate Alexa Fluor antibody (Cell Signaling Technology). Cells were counterstained with DAPI before observation. All antibodies were diluted according to product guidelines. Microscope images were acquired with a Leica DMI6000 B using Leica AF6000 software.
  • siRNAs in this study were purchased from GenePharma. The product guide, cells were transfected with GenePharmasiRNAMate TM (GenePharma) of siRNAs. The lentivirus used for YAP interference and overexpression was obtained from GenePharm. The lentivirus used for the alanine mutant GRs at S211 was purchased from Cyagen. Cells were selected with 5 ⁇ g/mL puromycin (AMRESCO) after lentiviral infection. The siRNA sequences used in this study were siGR#1, 5'-GGAGAUCAGACCUGUUGAUTT-3' and siGR#2, 5'-GGAGAUGACAACUUGACUUTT-3'.
  • shRNA sequences used in this study were shYAP#1, 5'-CTGGTCAGAGATACTTCTTAA-3' and shYAP#2, 5'-AAGCTTTGAGTTCTGACATCC-3'. This study used the siGNB2L1 sequence previously described by Yao F (43).
  • BMSCs with mild lysis buffer 150 mM NaCl, 50 mM HEPES pH 7.5, 50 mM NaF, 10 mM pyrophosphate, 10 mM glycerophosphate, 1.5 mM Na3VO4, 1 mM EDTA, 1 mM PMSF, 1% NP-40, protease inhibitor cocktail [Sigma- Aldrich]) dissolved.
  • the cell lysate was then centrifuged at 12,000 g for 15 minutes at 4 ° C, and the supernatant was separated to obtain an immunoprecipitation of endogenous LATS1 and MST1.
  • the supernatant was added to the LATS1 or MST1 antibody for 1 hour. After incubation with protein A-agarose beads for 90 minutes and three times with cell lysis buffer, the protein beads were washed once with wash buffer (40 mM HEPES, 200 mM NaCl), kinase assay buffer (30 mM HEPES, 50 mM potassium acetate, 5 mM MgCl2). Wash once. At 300 ⁇ M In the presence of cold ATP, endogenous MST1 was involved in the kinase assay and 1 ⁇ g of GST-Mob expressed and purified from Escherichia coli was used as a substrate.
  • wash buffer 40 mM HEPES, 200 mM NaCl
  • kinase assay buffer 30 mM HEPES, 50 mM potassium acetate, 5 mM MgCl2
  • the reaction mixture was incubated at 30 ° C for 30 minutes, stopped with loading buffer and added to SDS-PAGE.
  • the Lats1 kinase assay was similar, but GST-YAP was used as a substrate. Phosphorylation of GST-YAP at S127 was determined by immunoblotting with pYAP antibody.
  • Micro CT scan The samples used in this study were scanned by SkyScan 1178 (Bruker MicroCT). The image pixel size is set to 9 ⁇ m.
  • Statistical data such as BV/TV for each sample, was calculated using a CT analyzer (Bruker MicroCT). Three views of a typical sample for each group were generated using a DataViewer (Bruker MicroCT).
  • Glucocorticoid induces osteonecrosis by inhibiting the secretion of HIF-1 ⁇ and angiogenic factors
  • vascular endothelial growth factor vascular endothelial growth factor
  • HIF-1 ⁇ vascular endothelial growth factor
  • HIF-1 ⁇ and VEGF were inhibited with BAY87-2243 and axitinib, and osteonecrosis was successfully reproduced in rats.
  • BAY87-2243 or axitinib After receiving BAY87-2243 or axitinib, a typical osteonecrosis phenotype occurred in the femoral head of the rat, which was confirmed by micro-CT scan and pathological examination (Fig. 1G and Fig. 1H). Similar to the MPS group, most of the trabecular bones in the femoral head were replaced by adipose tissue after treatment with BAY87-2243 or axitinib.
  • BMSCs were subjected to microarray analysis with or without DEX treatment. It was noted that the Hippo-YAP signaling pathway target genes including ANKRD1, CTGF and CYR61 were upregulated after glucocorticoid stimulation (Fig. 2A). Therefore, it is hypothesized that the Hippo-YAP signaling pathway may be involved in glucocorticoid-induced HIF-1 ⁇ inhibition.
  • the mRNA level of the YAP target gene was first evaluated by quantitative polymerase chain reaction (qPCR) assay, and continuous results were obtained (Fig. 2B). Western blot analysis showed that glucocorticoid-induced dephosphorylation of YAP (Fig.
  • YAP is required for glucocorticoid-induced HIF-1 ⁇ inhibition
  • RNA level of HIF-1 ⁇ did not change after DEX treatment or YAP overexpression (Fig. S3), indicating that YAP may control HIF-1 ⁇ degradation.
  • YAP transcriptional coactivator properties it is hypothesized that YAP may increase transcription of one or more genes, further promoting HIF-1 ⁇ degradation.
  • candidate genes that increase HIF-1 ⁇ degradation were identified by searching the NCBI database (15-27). The expression of all candidate genes in DEX treatment and YAP overexpressing BMSCs was tested and it was found that GNB2L1 was the most up-regulated in both conditions (Fig. 3C and Fig. S4).
  • the protein level of bone tissue GNB2L1 increased after injection of MPS (Fig. 3D and Fig. S2). Knockdown of GNB2L1 blocked the inhibition of HIF-1 ⁇ in DEX-treated cells, suggesting that glucocorticoids enhance HIF-1 ⁇ degradation by enhancing the expression of GNB2L1 (Fig. 3E).
  • Glucocorticoid receptor increases LATS kinase activity
  • Glucocorticoid binds to the glucocortic
  • the hormone receptor (GR) stimulates intracellular signals (28), which is a member of the transcription factor nuclear hormone receptor superfamily.
  • GREs glucocorticoid response elements
  • GR located in the cytosol may inhibit YAP activity.
  • Expression of the S211A GR engineered gene which is always located in the cytoplasm, blocked YAP activation, proving our hypothesis (Fig. 4B).
  • wild-type GR overexpression did not inhibit YAP dephosphorylation by DEX treatment (Fig. 4C). Therefore, it is concluded that the presence of GR in the nucleus is responsible for the glucocorticoid-induced YAP activation.
  • a kinase assay is performed to determine which kinase is regulated by GR.
  • a MST1/2 substrate revealed no detectable effect of DEX on MST1 kinase activity.
  • MST1/2 phosphorylation at T183/T180 was not affected (Fig. 4D).
  • LATS1 kinase activity was measured and it was found that LATS1 kinase activity was inhibited by DEX treatment and GR knockout.
  • Expression of the S211A GR engineered gene increased LATS1 kinase activity even in the presence of DEX (Fig. 4E).
  • Angiotensin-converting enzyme (ACE) inhibitor or angiotensin II receptor blocker prevents glucocorticoid-induced osteonecrosis
  • Glucocorticoids increase the expression of angiotensin receptor 1 (AGTR1) in bone marrow mesenchymal stem cells. Silencing of angiotensin receptor 1 by siRNA can block YAP activation and angiogenesis inhibition by hormones. This suggests that hormone up-regulation of angiotensin receptor 1 is one of the important mechanisms of hormone-activated YAP and inhibition of angiogenesis. Therefore, it is speculated that blocking the function of angiotensin receptor 1 can prevent YAP activation and prevent the occurrence of osteonecrosis (Fig. 10). Therefore, we attempted to prevent femoral head necrosis using an angiotensin receptor inhibitor (ARB) and an angiotensin converting enzyme inhibitor (ACEI).
  • ARB angiotensin receptor inhibitor
  • ACEI angiotensin converting enzyme inhibitor
  • GR can increase LATS kinase activity and further phosphorylate YAP.
  • Both glucocorticoid and Hippo-YAP signaling are required for cell biological functions including proliferation, apoptosis and metabolism.
  • We believe that the interference between these two pathways is not limited to osteonecrosis. Multiple diseases may be associated with the interaction of GR-Hippo.

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Abstract

本申请涉及血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂在预防或治疗骨坏死的药物中的应用，尤其是是糖皮质激素诱导的骨坏死，该骨坏死是由YAP介导的HIF-1a的抑制所导致。

Description

降压药在预防和治疗骨坏死中的应用 技术领域
本申请涉及降压药，如血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂用于预防或治疗骨坏死；还涉及HIF-1a激动剂在制备预防或治疗骨坏死的药物中的应用
背景技术
骨组织自身病变是引起骨坏死最常见的原因。目前，学者普遍认为骨组织的再生修复能力障碍，血供障碍是骨坏死发生的重要机制。激素和酒精是最常见的引起上述病理生理过程的体外因素，也是目前我国骨坏死的最主要病因。
股骨头坏死的症状和体征多种多样，病痛出现的时间，发作的程度也不尽相同。而各种临床表现都不是股骨头坏死所特有的，许多髋关节疾患都可以发生，换句话说，难以通过患者的主观症状和临床检查做出股骨头坏死的诊断来。此外，早期X线片可没有阳性发现。随进展，于负重区出现骨小梁紊乱，中断，以后股骨头软骨下骨囊性变、夹杂硬化，X线片呈现新月征，方可明确股骨头坏死的诊断。因此，患者就诊时骨坏死常已到了较为严重的阶段，进一步增加了治疗难度。由于尚无有效的药物用于治疗股骨头坏死，股骨头坏死的治疗只能依赖手术治疗。早期可采取保留股骨头的手术，如：髓芯减压、带血管骨移植术等。晚期不可避免的情况下，需行人工关节置换术。但总体看来，手术疗法因其痛苦大、费用高、恢复期长、远期效果不能尽善尽美等而不被众多患者所接受。
预防股骨头坏死主要依靠去除病因。对于酗酒和肥胖的人来说，戒酒和减肥能有效预防股骨头坏死的发生，甚至能逆转极早期的病变。但对于需大剂量应用激素冲击和长期服用激素的患者来说，停用激素是不现实的。因此，我们迫切需要一种能有效预防激素引起的股骨头坏死的药物。但令人遗憾的是，目前尚无这样一种药物。
糖皮质激素被广泛用于治疗多种疾病，包括自身免疫性疾病，急性成淋巴性白血病，肾脏综合症和严重的炎症。然而，长期或大剂量施用糖皮质激素会导致健康方面的问题。骨坏死也被称为无血管的坏死，它是常见的几种严重并发症之一，是在糖皮质激素治疗之后由于血液向骨组织的供应存在障碍而发生。例如，在接受糖皮质激素治疗的系统性红斑狼疮(1,2)患者中骨坏死的发生率为9.5％-37％。在器官移植的患者中，骨坏死发生率为器官接受者的4.6-20％(3,5)。尽管很多理论对骨坏死的发病给予了假设，但据我们所知还没有研究有直接证据在动物模型上再现骨坏死以支持这些理论。
之前有报告显示糖皮质激素可以抑制人体末梢肺上皮细胞内的缺氧诱导因子1α(HIF-1α)和血管生成因子的合成(6)。鉴于糖皮质激素有抗血管原作用(7-9)以及骨坏死没有血管的特点，可以想象血管再生的破坏可能与骨坏死发病机理相关。在本研究中，第一次证明了抑制HIF-1α和血管生成因子可以导致大鼠骨坏死。然后我们研究了糖皮质激素抑制HIF-1α的机制，并发现糖皮质激素诱导YAP的激活和随后GNB2L1表达升高是抑制HIF-1α所必需的。此外，我们报道了糖皮质激素受体(GR)和Hippo-YAP信号之间的新型干扰(crosstalk)，此干扰是造成糖皮质激素诱导YAP激活的原因。
骨坏死的手术和非手术治疗方法远不能使人满意，这突出了有效预防的需求。基于在骨坏死发病机理中的发现，血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂可用来防止施用高剂量糖皮质激素的大鼠发生骨坏死。
发明内容
本申请主要内容为血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂的新作用，用于预防或治疗骨坏死。
根据本申请的一个方面，本申请涉及血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂在制备预防或治疗骨坏死的药物中的应用。
根据本申请的另一方面，本申请涉及一种预防或治疗骨坏死的方法，其包括将治疗有效量的血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂施用于有此需要的受试者。
根据本申请的一些具体实施方式，上述骨坏死是糖皮质激素诱导的骨坏死。
根据本申请的一些实施方式，上述骨坏死是由YAP介导的HIF-1a的抑制所导致。
根据本申请的一些实施方式，血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂是通过抑制YAP的激活而预防或治疗骨坏死。
根据本申请的一些实施方式，所述血管紧张素转换酶(ACE)抑制剂优选为卡托普利，血管紧张素Ⅱ受体阻断剂优选为氯沙坦。
根据本申请的又一方面，本申请涉及HIF-1a激动剂在制备预防或治疗骨坏死的药物中的应用。根据本申请的再一方面，本申请涉及使用HIF-1a激动剂来预防或治疗骨坏死的方法。药物治疗骨坏死的主要难点在于：由于骨坏死区存在血供障碍，促进骨修复的药物常难以到达坏死区域。针对这一情况，我们把改善坏死区血供作为治疗骨坏死的主要方向。
血管损伤会导致缺血缺氧。缺血缺氧组织内的细胞会上调自身HIF-1a，从而促进血 管新生。我们发现糖皮质激素会激活YAP蛋白来抑制体内的HIF-1a，从而抑制血管新生。这种抑制打破了体内的血管损伤和血管新生的动态平衡，最终导致了骨组织血供消失，进而引起了骨坏死。
针对这一发病机制，我们应用血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂，抑制YAP，从而增加血管新生，预防糖皮质激素引起的骨坏死。对于已经发生的坏死，我们通过血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂的促血管新生作用，促进坏死区的修复。
糖皮质激素诱导的骨坏死是由YAP介导的HIF-1a的抑制所导致，血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂通过抑制YAP的激活可预防骨坏死。
根据本申请的再一方面，本申请涉及YAP抑制剂在制备预防或治疗骨坏死的药物中的应用。
根据一些实施方式，所述YPA抑制剂选自卡托普利和氯沙坦。
长期或高剂量施用糖皮质激素会导致骨坏死，通常在股骨头发生骨坏死，从而导致关节损伤，运动受限。本文首次研究了糖皮质激素导致的骨坏死是通过HIF-1a和一些血管新生因素受到抑制所致。本申请鉴定了YAP(Hippo通路的效应器)作为糖皮质激素诱导的信号转导的下游介导子，表现出通过GNB2L1的表达而抑制HIF-1a的活性。本申请进一步展现在糖皮质激素受体和Hippo信号之间的干扰(cross talk)对糖皮质激素的对YAP的激活起作用。最后，本申请使用血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂(降低血清胆固醇水平)靶向YAP并成功预防施用糖皮质激素的大鼠的骨坏死。长期服用激素或需要大量使用激素的患者，同时使用血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂，可以起到预防和逆转早期坏死的作用。
附图说明
图1.糖皮质激素抑制HIF-1α以阻止血管生成因子分泌。(A)糖皮质激素诱导的骨坏死患者和健康受试者的未感染骨组织的溶解产物用HIF-1α和VEGF抗体点渍法分析(每组n＝24)。与健康受试者比较的蛋白质含量统计结果如图所示。**P<0.01对比健康受试者，学生t检验。(B)用或不用甲基强的松龙(MPS)处理的大鼠骨组织的溶解产物用HIF-1α和VEGF抗体免疫印迹法分析(每组n＝7)。与对照组比较的蛋白质含量统计结果如图所示。**P<0.01对比对照组，学生t检验。(C)肌肉内注射MPS后24小时，大鼠股骨头的骨组织IHC染色。HIF-1α和VEGF在骨髓细胞和成骨细胞中的表达 被抑制(OBs，箭头)。典型特征如示(n＝10)。比例尺，100μm。(D)BMSCs和OBs按照指示处理。细胞溶解物用指示抗体免疫印迹标记。(E)用1μM DEX处理24小时后，正常含氧量或低氧条件下，BMSCs培养物上清物中指示血管生成因子的浓度。*P<0.05对比正常氧含量。**P<0.01对比正常含氧量。##P<0.01对比低含氧量，学生t检验。图中每组四个柱从左至右依次代表正常含氧量、低含氧量、正常含氧量+DEX、低含氧量+DEX。(F)1μM DEX在正常含氧量或低含氧环境下刺激24小时后BMSCs中血管生成因子mRNA水平。*P<0.05对比正常含氧量。**P<0.01对比正常含氧量。##P<0.01对比低含氧量，学生t检验。图中每组四个柱从左至右依次代表正常含氧量、低含氧量、正常含氧量+DEX、低含氧量+DEX(G)接受BAY 87-2243(4mg/kg)和阿西替尼(Axitinib，20mg/kg)六个月后的大鼠发展成骨坏死。每组典型微-CT图像。每组骨相关参数包括骨容量/组织容量(BV/TV)，小梁数目(Tb.N)，厚度(Tb.Th)，和分离(Tb.Sp)的统计结果。**P<0.01，学生t检验。柱状图中的四个柱从左至右依次代表对照、MPS、BAY 87-2243和阿西替尼。(H)每组典型的病理学区域。在MPS，BAY 87-2243和阿西替尼组中，小梁骨丢失，并且被脂肪组织替代。
图2.糖皮质激素诱导YAP激活。(A)微阵列数据(每组重复三次)热图显示在DEX处理24小时后，三个YAP下游基因(ANKRD1，CTGF，Cyr61)上调，并且VEGF显著下调。(B)三个下游基因的激活是由qPCR试验确定的。**P<0.01对比0nM，学生t检验。图中每组四个柱从左至右依次代表0nMDEX、10nMDEX、100nMDEX和1000nMDEX时mRNA的表达水平。(C)YAP亚细胞定位由DEX处理后的BMSCs的免疫荧光监测。核定位可在100nM DEX处理3小时后观测到。比例尺，100μm。(D)BMSCs被四种不同浓度的DEX(0nM,10nM,100nM,1,000nM)刺激3小时。BMSCs中YAP去磷酸化反应与DEX浓度正相关。(E)肌肉内注射MPS后24小时，YAP在大鼠股骨头骨组织中聚集。骨组织溶解物用免疫印迹法分析(每组n＝7)。显示与对照组蛋白质含量比较的统计结果。**P<0.01对比对照组，学生t检验。(F)肌肉内注射MPS后24小时，YAP和CTGF在大鼠股骨头骨髓细胞和OBs(箭头)中聚集。显示典型图片(n＝10)。比例尺，100μm。
图3.糖皮质激素诱导HIF-1α信号通路抑制是由激活的YAP介导。(A)在正常含氧量或缺氧条件下培养感染YAP或者空白(empty)慢病毒BMSCs。在缺氧条件下YAP过度表达阻止HIF-1α聚集。(B)感染病毒shYAP或对照shRNA的BMSCs在缺氧条件下被1μM DEX激发。(C)YAP过度表达或100nM DEX处理BMSCs后对候选基因进行检测。在两个试验中GNB2L1大多上调。**P<0.01。同见图S3.(D)大鼠骨组织GNB2L1 的蛋白质水平由免疫印迹法分析(每组n＝7)。相对对照组蛋白质含量的统计结果如示。**P<0.01对比对照组，学生t检验。(E)敲除GNB2L1缓解(rescue)DEX处理的BMSCs中的HIF-1α和VEGF。BMSCs按照指示在缺氧条件处理。(F)相对于健康受试者，骨坏死患者骨组织中YAP和GNB2L1的蛋白质水平显著提高。用指示抗体点渍法分析未感染骨组织的溶解产物(每组n＝24)。相对于健康受试者的蛋白质含量统计结果如示。**P<0.01对比对照组，学生t检验。
图4.糖皮质激素诱导YAP的激活由GR和LATS1/2相互调节(cross-talk)。(A)用siRNA敲除BMSCs中GR上调ANKRD1，CTGF，Cyr61的表达。**P<0.01对照siCTL，学生t检验。图中每组柱中最左侧柱代表siCTL。(B)当BMSCs用100nM DEX处理时，S211A GR基因改造表达阻断YAP核定位。比例尺100μm。(C)野生型基因改造表达不会如同S211A GR，阻断DEX诱导的YAP去磷酸作用。(D)内生MST1由用或不用DEX处理(100nM，3小时)的细胞溶解产物免疫沉淀，并且MST1激酶活性用GTS-MOB1作为底物测定。与激酶活性相关的MST1/2磷酸化作用在DEX处理后没有变化。(E)DEX通过激活LATS1诱导YAP磷酸化作用。内生LATS1是由按指示处理BMSCs免疫沉淀的。DEX处理(100nM，3小时)或GRs敲除后，LATS1活性升高。然而，阻断DEX诱导的S211A GR基因改造表达增加了LATS1激酶活性。
图S1.患者和健康受试者骨组织中YAP，GNB2L1，HIF-1α和VEGF的蛋白质含量。直至样品获取日，所有病人均接受了医疗目的的糖皮质激素治疗。蛋白质含量通过点渍法检测。
图S2.MPS肌肉内注射MPS后骨组织中YAP，GNB2L1，HIF-1α和VEGF的蛋白质水平。MPS处理24小时后，用蛋白质印迹法评价YAP，GNB2L1，HIF-1α和VEGF的蛋白质水平。
图S3.DEX处理或YAP过度表达后HIF-1A的mRNA水平。DEX处理(100nM)或YAP过度表达后，BMSCs中的HIF-1A表达没有显著变化。
图S4.抑制HIF-1α的候选基因的mRNA水平。BMSCs中YAP过度表达或100nMDEX处理后，用qPCR评价候选基因的mRNA水平。
图S9.血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂预防和治疗骨坏死的机制示意图
用siRNA敲除BMSCs中AGTR1后发现激素所导致的YAP激活和血管新生抑制被逆转了。
图S10.在给予糖皮质激素后，BMSCs中的AGTR1显著上调。而用siRNA沉默BMSCs中的AGTR1，发现给予糖皮质激素后，BMSC中的HIF-1α和VEGF不再被抑制。这说明上调AGTR1是糖皮质激素抑制HIF-1α和VEGF，进而影响血管新生诱发骨坏死的重要机制。
图S11.大鼠如图所示给药一天后，检测骨组织中所示蛋白的浓度。激素剂量同前，ARB代表给予氯沙坦，剂量为1mg/kg，ACEI代表给予卡托普利，剂量为1mg/kg。给药方式为灌胃。在给予氯沙坦和卡托普利后，骨组织中的HIF-1α和VEGF水平较之单给激素组回到了接近正常的水平。这说明上述两种药物可以有效逆转糖皮质激素所引起的血管新生抑制。由于卡托普利的作用机制为减少血液中血管紧张素II的生成，静脉给予血管紧张素II后，HIF-1α和VEGF的含量又回到了单给激素组类似的水平。上述实验进一步证实，糖皮质激素通过AGTR1受体抑制骨组织内的血管新生。
图S12.显示ACEI和ARB两种药物可预防激素所致股骨头坏死的发生。
图S13.显示ACEI和ARB两种药物能显著降低股骨头坏死的发生率。ARB代表给予氯沙坦，剂量为1mg/kg，ACEI代表给予卡托普利，剂量为1mg/kg。造模方式同前，初次给予激素注射6周后处死，两种预防药物每天给予灌胃。如图所示，使用ACEI和ARB可以有效预防骨坏死的发生。
注：图1至图4以及图S3、S4和S8中的柱状图，其上方或右上方的图标框里的图标自上向下依次对应于柱状图中自左至右的柱。
具体实施方式
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合实施例，对本申请进行进一步的详细说明。应当理解，此处所描述的具体实施例或实施方式仅仅用以解释本申请，并不用于限定本申请。
材料和方法
患者信息。从2015年3月到2015年6月，共计24名诊断患有骨坏死的患者加入我们的研究，他们曾接受糖皮质激素治疗直至髋关节置换；24名患者因为股骨颈骨折接受髋关节置换作为对照组加入。在手术过程中获得未受影响区域的松质骨，并且立即分离得到蛋白质，然后储存于-80℃以利于将来分析。受试者的人口统计学特征如附录表1所示。
动物。我们给予八周大的SD大鼠肌肉内注射MPS(20mg/kg)，每星期前三天注射，持续三星期，以形成骨坏死动物模型。第一次甲基强的松龙注射后，每天以胃内灌注方式给予溶解于植物油中的血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂。所有大鼠在第一次甲基强的松龙注射后六星期死亡。对于短期刺激，大鼠在MPS(20mg/kg)注射前连续三天接受氯沙坦治疗。大鼠在MPS注射后24小时死亡。每天胃内灌注给予提示剂量的BAY87-2243和阿西替尼持续六个月，然后大鼠死亡。
细胞分离，培养和化学处理。BMSCs是从因严重创伤经历截肢的健康受试者(年龄，30-50岁)中分离的。简单来说，松质骨在手术时获得，并且首次用培养介质冲洗，然后转移到T75容器。BMSCs是由粘连反应(adherence)分离的。BMSCs培养于α-MEM(粒化)，在37℃和5％CO2条件下补充10％胎牛血清(FBS；Gibco)。以下化学品用于本研究：地塞米松(Sigma-Aldrich)，。对于缺氧刺激，细胞培养于包含有10％FBS的α-MEM，有或没有地塞米松存在，在37℃，5％CO2和1％O2条件下培养24小时。
免疫印迹法分析。在冰上用Cellytic M(Sigma-Aldrich)溶解细胞30分钟。细胞溶解产物在15,000×g离心15分钟，其上清物收集用于免疫印迹分析。免疫印迹试验按照标准操作方案SDS-PAGE和点渍法实施。所有图片用FluorChem M系统(蛋白质样品)采集。
酶联免疫吸附测定法。此研究所有ELISA配套元件从Boster BiologicalTechnology获得。ELISA试验依据产品指南进行。样品加入到96孔板在37℃保持90分钟，所述96孔板用相应配套元件中的抗体预先涂覆。然后，在37℃加入生物素结合抗体，60分钟，然后用TBS洗涤三次。加入ABC溶液后，板子用TBS洗涤五次。测定前，37℃加入TMB溶液少于30分钟，然后用TMB终止缓冲液结束反应。
微阵列技术。微阵列实验用
Figure PCTCN2017073118-appb-000001
PrimeViewTM人体基因表达阵列进行。在Bioconductor的‘affy’文件包实行稳健多阵列平均值(RMA)推导法应用于原始强度数据以实现芯片间标准化。然后执行‘limma’文件包选择差异表达基因(DEGs)。精选的基因表达值转化成Z-分值，然后基于欧氏距离和平均连锁分等级聚集用于生成热图。
定量实时PCR。收集细胞于Trizol Reagent(Thermo Fisher Scientific Inc.)用于总RNA提取。RNA完整性和数量由NanoDrop 2000分析。污染物DNA和反转录的移除在EasyScriptOneStepgDNA Removal和cDNA Synthesis SuperMix(Beijing TransGen Biotech Co.,Ltd)上进行。得到的cDNA合理稀释并用于qPCR反应，该反应用TransStart Tip Green qPCR SuperMix(Beijing TransGen Biotech Co.,Ltd)进行，使用ABI 7900HT(Applied  Biosystems)，并用SDS 2.4(Applied Biosystems)分析。每个实验至少实施三次。本研究中GAPDH和β-肌动蛋白作为持家基因用于结果标准化。人体样本PCR寡核苷酸序列报告于附录表2。
抗体。VEGF抗体从Boster Biological Technology获得。YAP1小鼠抗体和HIF-1α抗体从Wuxi UcallM Biotechnology获得。糖皮质激素受体，YAP兔，MST1兔，MST2，LATS1(C66B5)，β-肌动蛋白(8H10D10)，phospho-MOB1(Thr12)，phospho-MST1(Thr183)/MST2(Thr180)和phospho-YAP(Ser127)(D9W2I)抗体从Cell Signaling Technology获得。MST1小鼠抗体室内培养。所有抗体基于依据产品指南的具体试验稀释。
免疫荧光染色。BMSCs以合适的密度在盖玻片上培养，然后根据指示处理。在4％多聚甲醛固定15分钟后，将BMSCs用0.1％Triton X-100渗透15分钟并在PBS中用10％FBS阻隔1小时。然后在37℃加入初始抗体1小时，随后用PBS洗涤三次，每次五分钟。接着培育细胞30分钟，用分别的Alexa Fluor结合抗体(Cell Signaling Technology)。观察前将细胞用DAPI复染色。所有抗体根据产品指南稀释。显微镜图像用Leica DMI6000 B采集，其使用Leica AF6000软件。
转染，慢病毒感染和RNA干扰。本研究中所有siRNA是从GenePharma购得。根据产品指南，细胞用GenePharmasiRNAMateTM(GenePharma)的siRNAs进行转染。YAP干扰和过度表达所用的慢病毒从GenePharm获得。在S211处丙氨酸突变GRs所用的慢病毒从Cyagen购得。细胞在慢病毒感染后用5μg/mL嘌呤霉素(AMRESCO)选择。本研究所用siRNA序列是siGR#1,5’-GGAGAUCAGACCUGUUGAUTT-3’和siGR#2,5’-GGAGAUGACAACUUGACUUTT-3’。本研究所用shRNA序列是shYAP#1,5’-CTGGTCAGAGATACTTCTTAA-3’和shYAP#2,5’-AAGCTTTGAGTTCTGACATCC-3’。本研究所用siGNB2L1序列由Yao F先前描述(43)。
免疫沉淀和激酶试验。BMSCs用温和溶解缓冲液(150mMNaCl，50mM pH 7.5的HEPES，50mMNaF，10mM焦磷酸盐，10mM甘油磷酸，1.5mM Na3VO4，1mM EDTA，1mM PMSF，1％NP-40，蛋白酶抑制剂混合剂[Sigma-Aldrich])溶解。然后将细胞溶解液在12,000g，4℃离心15分钟，分离上清物得到内生LATS1和MST1的免疫沉淀。LATS1或MST1抗体加入上清物1小时。用蛋白质A-琼脂糖珠培育90分钟以及用细胞溶解缓冲液清洗三次后，蛋白珠用清洗缓冲液(40mM HEPES，200mMNaCl)洗涤一次，激酶试验缓冲液(30mM HEPES，50mM potassium acetate,5mM MgCl2)洗涤一次。在300μM 冷(cold)ATP存在下，内生MST1参与激酶试验，并且以从Escherichia coli中表达和纯化的1μgGST-Mob作为底物。反应混合液在30℃培育30分钟，用加样缓冲液终止，加到SDS-PAGE。Lats1激酶试验操作类似，但是GST-YAP用作底物。在S127处GST-YAP的磷酸化作用由用pYAP抗体的免疫印迹法确定。
微CT扫描。本研究所用样品由SkyScan1178(Bruker MicroCT)扫描。图像像素大小设置为9μm。统计数据，如每个样品的BV/TV，是用CT分析仪计算的(Bruker MicroCT)。每组的典型样品的三个视图是用DataViewer(Bruker MicroCT)生成的。
统计法。统计分析是用OriginPro 2015(OriginLab)操作的。数值表示成至少三次独立实验的平均值±SD。当使用两组之间非配对、双尾学生t检验时，认为P值小于0.05有统计学意义。
研究批准。所有动物实验都根据上海交通大学医学院IACUC批准的操作流程实施。本研究人体BMSCs的使用获得上海交通大学附属第六人民医院伦理委员会的批准，并且根据赫尔辛基宣言取得所有受试者的知情同意。
糖皮质激素通过抑制HIF-1α和血管生成因子的分泌诱导骨坏死
最初，用点渍法评价糖皮质激素诱导的骨坏死患者和健康受试者未被影响的骨组织中血管内皮生长因子(VEGF)和HIF-1α的蛋白水平。统计结果表明患者骨组织中HIF-1α和VEGF均比正常受试者下降(图1A和图S1)。而且，在大鼠肌肉内注射甲基强的松龙(MPS)后，骨组织中HIF-1α和VEGF的蛋白含量也得到了抑制(图1B和图S2)。骨组织免疫组化染色显示HIF-1α和VEGF主要由骨髓细胞和成骨细胞(OBs)表达，并且给予MPS会抑制表达(图1C)。因此，用地塞米松(DEX)在含氧量正常和含氧量低的条件下激活骨髓基质细胞(BMSCs)和OBs，找出HIF-1α和VEGF在DEX处理后显著的下游调节(图1D)。DEX处理的BMSCs中mRNA和其他血管生成因子的分泌水平也显著下降(图1E和图1F)。
为研究HIF-1α的抑制是否为骨坏死发展的原因，用BAY87-2243和阿西替尼抑制HIF-1α和VEGF，并成功在大鼠身上重现骨坏死。在接受BAY87-2243或阿西替尼后，大鼠股骨头发生了典型的骨坏死显型，这由微CT扫描和病理学检查确认(图1G和图1H)。与MPS组相似，在BAY87-2243或阿西替尼处理后，大多数股骨头里的小梁骨(trabecularbones)被脂肪组织替代。先前报道表明HIF-1α降解下降可以阻止糖皮质激素引起的骨坏死，说明抑制HIF-1α是糖皮质激素诱导骨坏死所需要的(10)。综上所述，这些结果显示糖皮质激素通过抑制HIF-1α和血管生成因子分泌诱导骨坏死。
糖皮质激素激活YAP
为研究糖皮质激素如何抑制HIF-1α，用或不用DEX处理，对BMSCs进行微阵列分析。注意到Hippo-YAP信号通路靶基因包括ANKRD1，CTGF和CYR61在糖皮质激素刺激后上调(图2A)。因此猜测Hippo-YAP信号通路可能介入糖皮质激素诱导的HIF-1α抑制作用。首次用定量聚合酶链反应(qPCR)试验评价YAP靶基因的mRNA水平，得到了连续的结果(图2B)。免疫印迹法分析显示糖皮质激素诱导YAP的脱磷酸作用(图2D)，导致降解下降及随之而来的核定位(11-13)。YAP的核定位通过与TEAD家族转录因子结合(14)增加基因转录，在免疫荧光试验中DEX处理后也被观察到(图2C)。然后对糖皮质激素对YAP的影响进行体外评价。MPS注射后，蛋白印迹法(图2E和图S2)显示骨组织中的YAP显著聚集。有趣的是，由于注射MPS，YAP和CTGF也通常会在骨髓细胞和成骨细胞中聚集(图2F)。
YAP是糖皮质激素诱导HIF-1α抑制作用所必需的
我们过度表达BMSCs中的YAP，以判定YAP是否参与糖皮质激素主导的HIF-1α调节，以及YAP是否在含氧量低的条件下导致抑制HIF-1α聚集(图3A)。敲除YAP完全阻断糖皮质激素引起的对VEGF和HIF-1α的抑制(图3B)。因此，得出结论YAP是糖皮质激素诱导HIF-1α抑制作用所必需的。
HIF-1α的mRNA水平在DEX处理或YAP过度表达后没有变化(图S3)，说明YAP可能控制HIF-1α降解。鉴于YAP转录辅激活因子属性，猜测YAP可能增加一个或多个基因的转录，进一步促进HIF-1α降解。通过检索NCBI数据库，找到十四个增加HIF-1α降解的候选基因(15-27)。测试所有候选基因在DEX处理和YAP过度表达BMSCs中的表达，发现GNB2L1是两种条件上调最大的(图3C和图S4)。骨组织GNB2L1的蛋白水平在注射MPS后上升(图3D和图S2)。敲除GNB2L1阻断HIF-1α在DEX处理细胞的抑制作用，这表明糖皮质激素通过加强GNB2L1的表达提高HIF-1α降解(图3E)。
鉴于HIF-1α在骨坏死发病机理中的重要作用，得出结论YAP的激活可能是糖皮质激素诱导骨坏死发展过程所必需的。为进一步确认，通过进行YAP和GNB2L1的免疫印迹试验，研究患者骨组织中YAP和GNB2L1是否增加。糖皮质激素诱导的骨坏死患者的YAP和GNB2L1的蛋白水平显著上升(图3F和图S1)。
糖皮质激素受体增加LATS激酶活性
接着我们寻求确认糖皮质激素信号提升YAP活性的机制。糖皮质激素结合于糖皮质 激素受体(GR)来激发细胞内的信号(28)，所述受体是转录因子细胞核激素受体总科(superfamily)中的一员。在糖皮质激素结合的时候，GR在Ser211的位置被磷酸化并且从细胞浆转移到细胞核以识别特殊DNA序列，术语称为糖皮质激素应答元件(GREs)，从而增强或抑制靶基因的转录(29)。显然，敲除GR导致YAP靶基因表达升高，这与用糖皮质激素处理相似(图4A)。因为糖皮质激素处理和敲除GR两者都导致细胞浆GR减少，我们假设GR位于细胞浆中可能抑制YAP活性。始终位于细胞浆的S211A GR改造基因的表达阻断了YAP激活，证明我们的假设(图4B)。相比之下，野生型GR过度表达没有因DEX处理抑制YAP脱磷酸化(图4C)。因此，推断GR位于细胞核是糖皮质激素诱导YAP激活的原因。
因为YAP由两个Hippo激酶MST1/2和LATS1/2调控，因而进行激酶试验来判定哪个激酶是由GR调控。体外评价Mob(一个MST1/2底物)磷酸化作用发现DEX对MST1激酶活性没有可检测的效应。同样地，DEX处理后，在T183/T180的MST1/2磷酸化作用没有受影响(图4D)。接着，测量LATS1激酶活性，发现LATS1激酶活性被DEX处理和GR敲除所抑制。S211A GR改造基因的表达提高LATS1激酶活性，即使有DEX存在(图4E)。
血管紧张素转换酶(ACE)抑制剂或血管紧张素Ⅱ受体阻断剂预防糖皮质激素诱导的骨坏死
糖皮质激素可以增加骨髓间充质干细胞血管紧张素受体1(AGTR1)的表达。而将血管紧张素受体1通过siRNA的方法加以沉默可以阻断激素所导致的YAP激活和血管新生抑制。这说明激素上调血管紧张素受体1是激素激活YAP和抑制血管新生的重要机制之一。因此，推测阻断血管紧张素受体1的功能可以预防YAP激活并预防骨坏死的发生(图S10)。因此，我们尝试使用血管紧张素受体抑制剂(ARB)和血管紧张素转化酶抑制剂(ACEI)来预防股骨头坏死。我们发现上述两种药物可以有效阻断激素所导致的骨组织YAP激活和血管新生抑制(图S11)。最后，我们使用上述两种药物ACEI和ARB成功预防了激素所致股骨头坏死的发生(图S12)，并明显降低了股骨头坏死的坏死率，坏死率可由未使用这两种药的90％以上降低至10％左右(图S13)。
讨论
在现有研究中，结果支持骨坏死发病机理中长期存在的血管再生中断理论。骨坏死发病机理尚未明确。通常认为骨微循环中断会引起细胞死亡和骨结构破坏。当骨组织失 去血液供应，缺血性骨髓中的BMSCs和其他固有细胞(resident cell)分泌血管生成因子，包括VEGF，促进内皮细胞迁移及增强血管生成。我们的研究表明血管生成由用糖皮质激素治疗导致HIF-1α抑制和血管生成因子分泌而破坏。血管生成下降引发营养血管数目下降，最终导致骨坏死。
糖皮质激素如何抑制HIF-1α的机制尚不知晓。因此我们进行了地塞米松处理的细胞的微阵列分析，并且证明了YAP通过提高GNB2L1表达，使糖皮质激素诱导HIF-1α抑制。有趣的是，YAP的激活也可以在特定细胞群体自发诱导凋亡(38-41)。与HIF-1α不相关的机制可能也存在于YAP促进骨坏死的进程中。阐述这个重要问题需要更深入的研究。
显然，我们的结果也解释了GR可以增加LATS激酶活性，并且进一步磷酸化YAP。糖皮质激素和Hippo-YAP信号转导均为细胞生物学功能包括增殖，凋亡和代谢所必需的。我们认为这两个通路之间的干扰不限于骨坏死。多种疾病可能与GR-Hippo互相影响相关。

Claims (7)

  1. 血管紧张素转换酶抑制剂或血管紧张素II受体阻断剂在制备预防或治疗骨坏死的药物中的应用。
  2. 根据权利要求1所述的应用，其中所述的骨坏死是糖皮质激素诱导的骨坏死。
  3. 根据权利要求1或2所述的应用，其中所述骨坏死是由HIF-1a的抑制所导致。
  4. 根据权利要求3所述的应用，其中所述骨坏死是由YAP介导的HIF-1a的抑制所导致。
  5. 根据权利要求1至4任一项所述的应用，其中血管紧张素转换酶(ACE)抑制剂是卡托普利，血管紧张素Ⅱ受体阻断剂是氯沙坦。
  6. 根据权利要求1至5任一项所述的应用，其中所述血管紧张素Ⅱ受体阻断剂的有效剂量为1mg/kg-4mg/kg，血管紧张素转换酶(ACE)抑制剂的有效剂量为1mg/kg-5mg/kg。
  7. HIF-1a激动剂在制备预防或治疗骨坏死的药物中的应用。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1377259A (zh) * 1999-04-30 2002-10-30 千年药品公司 抑制ace－2的化合物及其使用方法
CN101632678A (zh) * 2009-09-01 2010-01-27 严洁 一种氯沙坦钾氢氯噻嗪组合物及其制备方法
US20130022676A1 (en) * 2010-03-05 2013-01-24 University Of Strathclyde Pulsatile drug release

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1377259A (zh) * 1999-04-30 2002-10-30 千年药品公司 抑制ace－2的化合物及其使用方法
CN101632678A (zh) * 2009-09-01 2010-01-27 严洁 一种氯沙坦钾氢氯噻嗪组合物及其制备方法
US20130022676A1 (en) * 2010-03-05 2013-01-24 University Of Strathclyde Pulsatile drug release

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WO2018145268A1 (zh) 降压药在预防和治疗骨坏死中的应用

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