Histamine H1 receptor inverse agonists improve structure and pain in an osteoarthritis mouse model

Abstract

Osteoarthritis (OA) is the most common joint disease. Controlling the complex pathogenesis is challenging, thus, disease-modifying OA drugs are not available. Forkhead box O (FOXO) transcription factors contribute to cartilage homeostasis through autophagy and oxidative stress resistance. Here, we sought to discover FOXO activators and found that cyproheptadine, a histamine H1 receptor (HRH1) inverse agonist, promoted FOXO3 nuclear translocation and increased FOXO target genes while suppressing inflammation. In a murine OA model, cyproheptadine reduced structural joint tissue damage and pain behaviors. Mechanistically, the inhibition of HRH1 constitutive activity mediated the effects of cyproheptadine on calcium balance between endoplasmic reticulum (ER) and cytoplasm, and FOXO activation was part of this mechanism. The antiinflammatory effect of cyproheptadine involved the inhibition of protein kinase C/NF-κB pathway. HRH1 inhibition also suppressed osteogenesis in mesenchymal stem cells and nerve growth factor expression, which are mechanisms of osteophyte formation and pain behaviors. Moreover, cyproheptadine suppressed ER stress-induced lipogenesis by upregulating insulin-induced gene 1. Our findings suggest that HRH1 constitutive activity controls important OA-promoting mechanisms and indicate that HRH1 inverse agonists are promising drug repurposing candidates for structure and pain improvement in OA.

Keywords: Aging; Bone biology; Calcium signaling; Cartilage; Cell biology; Osteoarthritis.

Figure 1. Effects of cyproheptadine in human chondrocytes.

FOXO1 (A) and FOXO3 (B) localization in human chondrocytes (n = 3) 24 hours after treatment with DMSO or cyproheptadine (CYP) (30 μM) was analyzed by immunocytochemistry. Scale bar: 50 μm. Nuclear color intensity of FOXO1 and FOXO3 was quantified. Human chondrocytes (n = 6) were incubated with the indicated doses of CYP for 24 hours and RNA was isolated for qRT-PCR for FOXO1 and FOXO3 genes (C) and FOXO target genes (D). (E) Relative mRNA levels of IL6 and MMP13 in human chondrocytes (n = 6) incubated with IL-1β (1 ng/mL) for 6 hours after pretreatment with or without the indicated doses of CYP for 24 hours in qRT-PCR. Data are presented as means ± SD. Statistical analysis in A and B was performed using Student’s t test. Statistical analysis in C and D was performed using one-way ANOVA with the Dunnett’s post hoc test. Statistical analysis in E was performed using 1-way ANOVA with Tukey-Kramer post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 2. Effects of cyproheptadine on structural changes in mice with experimental OA.

(A) Mice were treated with cyproheptadine (low dose: 5mg/kg, high dose: 10mg/kg) or control vehicle after DMM surgery. Representative Safranin-O staining images of whole joint (B), medial cartilage (C), synovium (D), and osteophyte (E). Scale bar: 300 μm. OARSI score of medial femoral condyle and tibial plateau (F), synovitis score (G), and osteophyte maturity score (H) following 12 weeks of cyproheptadine treatment. Sham vehicle (n = 14), DMM vehicle (n = 13), DMM CYP (low) (n = 15), and DMM CYP (high) (n = 14). Immunohistochemistry of FOXO3 (n = 6) (I) and IL-6 (n = 7) (J) in cartilage of mice with sham or DMM surgery treated with vehicle or CYP. Scale bar: 100 μm. Data are presented as means ± SD. Statistical analysis in F–H was performed using 1-way ANOVA with the Dunnett’s post hoc test. Statistical analysis in I and J was performed using 1-way ANOVA with the Tukey-Kramer post hoc test. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 3. Transcriptomic changes induced by cyproheptadine in human chondrocytes.

RNA-seq was performed on human chondrocytes (n = 5) treated with cyproheptadine (CYP) (30 μM) or control vehicle (DMSO) for 24 hours. For IL-1β stimulation, chondrocytes (n=5) were incubated with IL-1β (1 ng/mL) for 6 hours following the pretreatment with cyproheptadine (30 μM) for 24 hours. (A) Principal component analysis showing separation in 4 groups. (B) Volcano plot of the differentially expressed genes (DEGs) in CYP versus control. Metascape enrichment (C) and TRRUST (D) analysis using the upregulated genes after CYP treatment. (E) Gene set enrichment analysis (GSEA) showing enrichment of “autophagy” in chondrocytes treated with CYP. (F) Western blot analysis of LC3 in chondrocytes (n = 5) incubated with chloroquine (CQ) (25 μM) for 2 hours after pretreatment with or without CYP (30 μM) for 24 hours. Data are presented as means ± SD. Statistical analysis was performed using 1-way ANOVA with the Tukey-Kramer post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4. Antiinflammatory effects of cyproheptadine via NF-κB pathway.

(A) Volcano plot of the DEGs in RNA-seq performed on human chondrocytes (n = 5) treated with IL-1β (1 ng/mL) for 6 hours. (B) Volcano plot of the DEGs in RNA-seq performed on human chondrocytes (n = 5) treated with IL-1β (1 ng/mL) for 6 hours after pretreatment with or without cyproheptadine (CYP) for 24 hours. (C) Venn diagram of the shared upregulated genes by IL-1β stimulation and the downregulated genes by CYP treatment under IL-1β stimulation. Metascape enrichment (D) and TRRUST (E) analysis using the shared genes in C. (F) GSEA showing induced “cytokine-mediated signaling pathway” in chondrocytes treated with IL-1β. (G) GSEA showing inhibited “cytokine-mediated signaling pathway” in chondrocytes treated with CYP under IL-1β stimulation. (H) Western blot analysis of total p65 and phosphorylated p65 at Ser536 (p-p65 (Ser536)) in chondrocytes (n = 4) incubated with IL-1β (1 ng/mL) for 20 minutes after pretreatment with or without CYP (30 μM) for 24 hours. (I) Immunocytochemistry of p65 in chondrocytes (n = 3) incubated with IL-1β (1 ng/mL) for 20 minutes after pretreatment with or without CYP (30 μM) for 24 hours. Scale bar: 50 μm. Data are presented as means ± SD. Statistical analysis was performed using 1-way ANOVA with the Tukey-Kramer post hoc test. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 5. Cyproheptadine signaling via histamine H1 receptor.

(A) Transcripts per kilobase million (TPM) in RNA-seq of human chondrocytes (n = 5) for histamine receptors. (B) Relative mRNA levels of IL6 and MMP13 in human chondrocytes (n = 6) incubated with histamine (10 μM) for 6 hours after pretreatment with cyproheptadine (CYP) (30 μM) for 24 hours. (C) IHC of HRH1 in human normal and OA cartilage. n = 6. Scale bar: 100 μm. (D) Immunocytochemistry of FOXO3 in human chondrocytes (n = 3) transfected with siCtrl or siHRH1. Scale bar: 50 μm. (E) Relative mRNA levels of HRH1 and FOXO target genes in chondrocytes (n = 6) transfected with siCtrl or siHRH1. Human chondrocytes were incubated with histamine (10 μM) (n = 6) (F) or IL-1β (1 ng/mL) (n = 5) (G) for 6 hours after siRNA transfection, and RNA was isolated for qRT-PCR for IL6 and MMP13 genes. Data are presented as means ± SD. Statistical analysis in B, F, and G was performed using 1-way ANOVA with the Tukey-Kramer post hoc test. Statistical analysis in C–E was performed using Student’s t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 6. Cyproheptadine regulation of calcium signaling.

(A) GSEA showing inhibited “calcium-mediated signaling” in chondrocytes treated with cyproheptadine (CYP). TPM in RNA-seq of human chondrocytes (n = 5) for ITPRs (B), RYRs (C), and ATP2As (D). (E) Overview of compound effects on intracellular calcium dynamics via HRH1 signaling. (F) Intracellular calcium levels in TC28 cells (n = 3) following histamine (10 μM) stimulation after pretreatment with DMSO or cyproheptadine (CYP) (30 μM) for 1 hour. (G) Intracellular calcium levels in TC28 cells (n = 3) with DMSO or CYP treatment. (H) Intracellular calcium levels in TC28 cells (n = 3) following thapsigargin (1 μM) stimulation after pretreatment with DMSO or CYP for 1 hour. Data are presented as means ± SD. Statistical analysis was performed using Student’s t test. *P < 0.05, ***P < 0.001.

Figure 7. Mechanisms of cyproheptadine effects on the FOXO/autophagy axis via calcium signaling.

(A) Overview of compound effects on intracellular calcium dynamics. (B) Western blot analysis of total AKT and phosphorylated AKT at Ser473 (p-AKT (Ser473)) in chondrocytes (n = 4) incubated with ionomycin (IONO) (0.2 μM), thapsigargin (Tg) (1 μM), or Yoda1 (10 μM) for 30 minutes. (C) Intracellular calcium levels in TC28 cells (n = 3) following IONO (0.2 μM) stimulation with DMSO or CYP (30 μM). (D) Western blot analysis of total AKT and p-AKT (Ser473) in human chondrocytes (n = 3) incubated with DMSO, CYP (30 μM), CYP and IONO (0.2 μM), BEZ235 (0.1 μM), or BEZ235 and IONO for 30 minutes. (E) Immunocytochemistry of FOXO3 in human chondrocytes (n = 3) 24 hours after treatment with CYP and IONO. Scale bar: 50 μm. (F) Relative mRNA levels of FOXO target genes in chondrocytes (n = 5) treated with CYP and IONO. Data are presented as means ± SD. Statistical analysis in B and C was performed using Student’s t test. Statistical analysis in D–F was performed using 1-way ANOVA with the Tukey-Kramer post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 8. Mechanisms of cyproheptadine antiinflammatory effects via protein kinase C pathway.

(A) Relative mRNA levels of IL6 and MMP13 in human chondrocytes (n = 6) incubated with IL-1β (1 ng/mL) for 6 hours after pretreatment with or without cyproheptadine (CYP) and ionomycin (IONO). (B) Diagram of HRH1 signaling pathway. (C) Relative mRNA levels of IL6 and MMP13 in human chondrocytes (n = 7) incubated with IL-1β (1 ng/mL) for 6 hours after pretreatment with or without CYP and phorbol 12-myristate 13-acetate (PMA) (20 nM). (D) Western blot analysis of total p65 and phosphorylated p65 at Ser536 (p-p65 (Ser536)) in chondrocytes (n = 3) incubated with PMA (20 nM) for 20 minutes after pretreatment with or without CYP (30 μM) for 24 hours. (E) Immunocytochemistry of p65 in chondrocytes (n = 3) incubated with PMA (20 nM) for 40 minutes after pretreatment with or without CYP (30 μM) for 24 hours. Scale bar: 50 μm. Data are presented as means ± SD. Statistical analysis was performed using 1-way ANOVA with the Tukey-Kramer post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 9. Effects of cyproheptadine on osteogenesis.

(A) Alkaline phosphatase (ALP) staining in human mesenchymal stem cells (MSCs) incubated in growth medium or osteogenic medium in the presence or absence of IL-1β (1 ng/mL) for 7 days. (B) Relative mRNA levels of HRH1 in MSCs (n = 4) incubated in growth medium or osteogenic medium in the presence or absence of IL-1β (1 ng/mL) for 7 days. (C) Alizarin red S staining in MSCs incubated in growth medium or osteogenic medium in the presence or absence of IL-1β (1 ng/mL) for 28 days. ALP staining (D) and relative ALP activity (E) in MSCs (n = 4) incubated in growth medium or osteogenic medium in the presence or absence of CYP (5, 10, 20, or 30 μM) and IL-1β (1 ng/mL) for 7 days. Alizarin red S (ARS) staining (F) and its relative quantification (G) in MSCs (n = 4) incubated in growth medium or osteogenic medium in the presence or absence of CYP (5, 10, 20 or 30 μM) and IL-1β (1 ng/mL) for 28 days. Data are presented as means ± SD. Statistical analysis was performed using 1-way ANOVA with the Dunnett’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 10. Effects of cyproheptadine on pain behaviors.

Pain behaviors were evaluated by von Frey test (A) and pressure application measurement (PAM) (B) in mice treated with cyproheptadine (CYP) (low dose, 5 mg/kg; high dose, 10 mg/kg) or control vehicle after DMM surgery (Figure 2A). DMM vehicle (n = 13), DMM CYP (low) (n = 15), and DMM CYP (high) (n = 14). (C) GSEA showing induced “regulation of neurogenesis” in chondrocytes treated with IL-1β. (D) Enriched GO: 0050767 “regulation of neurogenesis” in the upregulated DEGs by IL-1β and the downregulated DEGs by CYP with IL-1β in RNA-seq. (E) Venn diagram and heat map of the shared genes in GO: 0050767 “regulation of neurogenesis”, the upregulated DEGs by IL-1β and the downregulated DEGs by CYP with IL-1β. Statistical analysis was performed using 1-way ANOVA with the Dunnett’s post hoc test. **P < 0.01, ***P < 0.001, ****P < 0.0001.

Figure 11. Cyproheptadine regulation of ER stress-induced lipid/cholesterol biosynthesis.

(A) Heat map of fatty acid and cholesterol biosynthesis-related genes in RNA-seq in human chondrocytes 24 hours after treatment with cyproheptadine (CYP) (30 μM). (B) Diagram of 3 major regulators for lipid/cholesterol biosynthesis, SREBP1/2, SCAP, and INSIG1. Time courses of INSIG1, SCAP, SREBF1, and SREBF2 in chondrocytes (n = 3) incubated with CYP (30 μM) (C) or thapsigargin (Tg) (1 μM) (D) for 24 hours in qRT-PCR. (E) BODIPY staining (n = 3) in chondrocytes incubated with CYP, Tg, or CYP and Tg for 24 hours. Scale bar: 50 μm. (F) IHC of INSIG1 in human normal and OA cartilage. n = 5. Scale bar: 100 μm. (G) IHC of INSIG1 in cartilage of mice with sham or DMM surgery treated with vehicle or CYP. n = 7. Scale bar: 100 μm. Data are presented as means ± SD. Statistical analysis in E and G was performed using 1-way ANOVA with the Tukey-Kramer post hoc test. Statistical analysis in F was performed using the Mann-Whitney test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.