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Isorhamnetin attenuates osteoarthritis by inhibiting osteoclastogenesis and protecting chondrocytes through modulating reactive oxygen species homeostasis

Zhou, Feng, Mei, Jingtian, Yuan, Kai, Han, Xiuguo, Qiao, Han, Tang, Tingting·2019·Journal of Cellular and Molecular Medicine
DOI 10.1111/jcmm.14333PMC PMC6533508
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Abstract

Increasing evidence indicates that osteoarthritis (OA) is a musculoskeletal disease affecting the whole joint, including both cartilage and subchondral bone. Reactive oxygen species (ROS) have been demonstrated to be one of the important destructive factors during early‐stage OA development. The objective of this study was to investigate isorhamnetin (Iso) treatment on osteoclast formation and chondrocyte protection to attenuate OA by modulating ROS. Receptor activator of nuclear factor‐kappa B ligand (RANKL) was used to establish the osteoclast differentiation model in bone marrow macrophages (BMMs) in vivo. H 2 O 2 was used to induce ROS, which could further cause chondrocyte apoptosis. We demonstrated that Iso suppressed RANKL‐induced ROS generation, which could mediate osteoclastogenesis. Moreover, we found that Iso inhibited osteoclast formation and function by suppressing the expression of osteoclastogenesis‐related genes and proteins. We proved that Iso inhibited RANKL‐induced activation of mitogen‐activated protein kinase activation of mitogen‐activated protein kinase (MAPK), nuclear factor‐kappa B (NF‐κB) and AKT signalling pathways in BMMs. In addition, Iso inhibited ROS‐induced chondrocyte apoptosis by regulating apoptosis‐related proteins. Moreover, Iso was administered to an anterior cruciate ligament transection (ACLT)‐induced OA mouse model. The results indicated that Iso exerted beneficial effects on inhibiting excessive osteoclast activity and chondrocyte apoptosis, which further remedied cartilage damage. Overall, our data showed that Iso is an effective candidate for treating OA.

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Figure 1 Iso inhibits receptor activator of nuclear factor‐kappa B ligand (RANKL)‐induced reactive oxygen species (ROS) production and osteoclastogenesis in vitro. (A) The chemical structure of Iso. (B) Bone marrow macrophages (BMMs) were treated with various concentrations of Iso for 24 h and analysed using a CCK‐8 assay. (C) Intracellular ROS levels of BMMs detected by DCFH‐DA probe. (D) Tartrate‐resistant acid phosphatase staining of osteoclasts treated with different concentrations of Iso. (E) Representative SEM images of bone resorption pits. *Compared with the control group and #compared with the RANKL and M‐CSF without Iso group
Figure 1 Iso inhibits receptor activator of nuclear factor‐kappa B ligand (RANKL)‐induced reactive oxygen species (ROS) production and osteoclastogenesis in vitro. (A) The chemical structure of Iso. (B) Bone marrow macrophages (BMMs) were treated with various concentrations of Iso for 24 h and analysed using a CCK‐8 assay. (C) Intracellular ROS levels of BMMs detected by DCFH‐DA probe. (D) Tartrate‐resistant acid phosphatase staining of osteoclasts treated with different concentrations of Iso. (E) Representative SEM images of bone resorption pits. *Compared with the control group and #compared with the RANKL and M‐CSF without Iso groupCC BY · PubMed Central
Figure 2 Iso suppresses receptor activator of nuclear factor‐kappa B ligand (RANKL)‐induced expression of osteoclastogenesis‐related genes and proteins. The expression of osteoclast‐specific mRNA, including (A) NFATc1, (B) tartrate‐resistant acid phosphatase, (C) c‐FOS, (D) DC‐STAMP, (E) cathepsin K, and (F) MMP‐9, was analysed using qRT‐PCR. (G) Western blotting was performed to analyse the expression of osteoclastogenesis‐related transcription factors including NFATc1 and c‐FOS. (H) Quantification of NFATc1 and c‐FOS expression levels. *Compared with the control group, and #compared with the RANKL and M‐CSF without Iso group
Figure 2 Iso suppresses receptor activator of nuclear factor‐kappa B ligand (RANKL)‐induced expression of osteoclastogenesis‐related genes and proteins. The expression of osteoclast‐specific mRNA, including (A) NFATc1, (B) tartrate‐resistant acid phosphatase, (C) c‐FOS, (D) DC‐STAMP, (E) cathepsin K, and (F) MMP‐9, was analysed using qRT‐PCR. (G) Western blotting was performed to analyse the expression of osteoclastogenesis‐related transcription factors including NFATc1 and c‐FOS. (H) Quantification of NFATc1 and c‐FOS expression levels. *Compared with the control group, and #compared with the RANKL and M‐CSF without Iso groupCC BY · PubMed Central
Figure 3 Iso treatment prevents osteoclastogenesis by regulating mitogen‐activated protein kinase (MAPK), NF‐κB and AKT signalling pathways. (A) The effects of Iso on MAPK signalling pathway proteins, including p‐ERK/ERK, p‐JNK/JNK and p‐p38/p38. (B) Western blotting showed that Iso affected the NF‐κB pathway‐related proteins IκBα, p65 and p‐p65. (C) The effects of Iso on AKT and the phosphorylation of AKT. *Compared with the control group and #compared with the receptor activator of nuclear factor‐kappa B ligand and M‐CSF without Iso group. (D) A schematic illustration showing signalling pathways involved in osteoclast differentiation regulated by Iso
Figure 3 Iso treatment prevents osteoclastogenesis by regulating mitogen‐activated protein kinase (MAPK), NF‐κB and AKT signalling pathways. (A) The effects of Iso on MAPK signalling pathway proteins, including p‐ERK/ERK, p‐JNK/JNK and p‐p38/p38. (B) Western blotting showed that Iso affected the NF‐κB pathway‐related proteins IκBα, p65 and p‐p65. (C) The effects of Iso on AKT and the phosphorylation of AKT. *Compared with the control group and #compared with the receptor activator of nuclear factor‐kappa B ligand and M‐CSF without Iso group. (D) A schematic illustration showing signalling pathways involved in osteoclast differentiation regulated by IsoCC BY · PubMed Central
Figure 4 Iso inhibits reactive oxygen species (ROS)‐induced chondrocyte apoptosis. (A) The production of intracellular ROS detected by DCFH‐DA probe. (B) Primary chondrocyte apoptosis assessed using flow cytometry analysis. (C) Measurement of mitochondrial membrane potential by JC‐1. (D) TUNEL staining to assess cell apoptosis. *Compared with the control group and #compared with the H2O2 without Iso group
Figure 4 Iso inhibits reactive oxygen species (ROS)‐induced chondrocyte apoptosis. (A) The production of intracellular ROS detected by DCFH‐DA probe. (B) Primary chondrocyte apoptosis assessed using flow cytometry analysis. (C) Measurement of mitochondrial membrane potential by JC‐1. (D) TUNEL staining to assess cell apoptosis. *Compared with the control group and #compared with the H2O2 without Iso groupCC BY · PubMed Central
Figure 5 Iso inhibits chondrocyte apoptosis by modulating apoptosis‐related genes and proteins. (A) The expression of apoptosis‐related genes, including Bcl‐xL/Bcl‐2 (anti‐apoptotic) and Bax/Bad (pro‐apoptotic), was evaluated using qRT‐PCR. (B) Western blotting showed the effects of Iso on apoptosis‐related proteins Bcl‐2 and Bax. *Compared with the control group and # compared with the H2O2 without Iso group
Figure 5 Iso inhibits chondrocyte apoptosis by modulating apoptosis‐related genes and proteins. (A) The expression of apoptosis‐related genes, including Bcl‐xL/Bcl‐2 (anti‐apoptotic) and Bax/Bad (pro‐apoptotic), was evaluated using qRT‐PCR. (B) Western blotting showed the effects of Iso on apoptosis‐related proteins Bcl‐2 and Bax. *Compared with the control group and # compared with the H2O2 without Iso groupCC BY · PubMed Central
Figure 6 Micro‐CT analysis of Iso‐treated OA induced by anterior cruciate ligament transection (ACLT). (A) Three‐dimensional μCT images of frontal views of the knee joints at 4 wk after the sham operation or the ACLT operation. (B) Sagittal views of the medial compartment of subchondral bone. (C) Quantitative analysis of BV, BV/TV, Tb.N, Tb.Sp and Tb.Th. *Compared with the control group and #compared with the ACLT group
Figure 6 Micro‐CT analysis of Iso‐treated OA induced by anterior cruciate ligament transection (ACLT). (A) Three‐dimensional μCT images of frontal views of the knee joints at 4 wk after the sham operation or the ACLT operation. (B) Sagittal views of the medial compartment of subchondral bone. (C) Quantitative analysis of BV, BV/TV, Tb.N, Tb.Sp and Tb.Th. *Compared with the control group and #compared with the ACLT groupCC BY · PubMed Central
Figure 7 Staining analysis of Iso treatment in anterior cruciate ligament transection (ACLT)‐induced osteoarthritis. Knee joint medial compartment cartilage and subchondral bone were stained by (A) S&F and (B) H&E. (C) Osteoclasts in subchondral bone were stained by tartrate‐resistant acid phosphatase. (D‐E) TUNEL and MMP‐3 staining of chondrocytes in articular cartilage. (F) Quantitative analysis of the Osteoarthritis Research Society International score and positively stained cells in articular cartilage. *Compared with the control group and #compared with the ACLT group
Figure 7 Staining analysis of Iso treatment in anterior cruciate ligament transection (ACLT)‐induced osteoarthritis. Knee joint medial compartment cartilage and subchondral bone were stained by (A) S&F and (B) H&E. (C) Osteoclasts in subchondral bone were stained by tartrate‐resistant acid phosphatase. (D‐E) TUNEL and MMP‐3 staining of chondrocytes in articular cartilage. (F) Quantitative analysis of the Osteoarthritis Research Society International score and positively stained cells in articular cartilage. *Compared with the control group and #compared with the ACLT groupCC BY · PubMed Central

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Metadata

Authors
Zhou, F., Mei, J., Yuan, K., Han, X., Qiao, H., & Tang, T.
Affiliations
Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedic Surgery Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine Shanghai P. R. of China
ORCID
Tang, Tingting
Venue
Journal of Cellular and Molecular Medicine
Publisher
Wiley
Published
2019-04-14
Volume
23
Issue
6
Pages
4395-4407
ISSN
  • 1582-1838
  • 1582-4934
DOI
10.1111/jcmm.14333
PMC
PMC6533508
Cited by
57
References
1
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)
Funders
  • National Natural Science Foundation of China (81672205)
  • Shanghai Science and Technology Development Foundation (18DZ2291200, 18441902700)

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