Cissus quadrangularis inhibits IL-1β induced inflammatory responses on chondrocytes and alleviates bone deterioration in osteotomized rats via p38 MAPK signaling

Figures & data

Figure 1 Morphological changes of chondrocytes and chondroprotective activity of herbals.

Notes: (A) Osteoarthritic chondrocytes grown in monolayer tend to attain a fibroblastic morphology. (B) Incubation with culture media containing TGF-β for 10–12 days induce redifferentiation to chondrocytes confirmed by the spherical morphology. (C) LDH release was used to determine cytotoxicity effects induced by the herbal extracts and (D) cell viability analyzed using CyQUANT assay. Figure shows representative data of three independent experiments run in triplicate. Values correspond to means ± standard error; *P<0.05. “−” means the IL-1β was not added to the treatment whereas “+” denotes that IL-1β was added before herbal treatment.
Abbreviations: C. quad, Cissus quadrangularis; IL-1β, interleukin-1β; LDH, lactate dehydrogenase; TGF-β, transforming growth factor-β; W. som, Withania somnifera.

Figure 2 Regulation of nitric oxide and glycosaminoglycan production with herbal treatments.

Notes: IL-1β pretreated chondrocytes were incubated with increasing concentrations of herbal sample for 24 hours. Conditioned media was collected and measured for the production of NO (A) and GAG (B). Figure shows representative data of two independent experiments run in triplicate and corresponds to means ± standard error; *P<0.05; **P<0.01. “−” means the IL-1β was not added to the treatment whereas “+” denotes that IL-1β was added before herbal treatment.
Abbreviations: C. quad, Cissus quadrangularis; GAG, glycosaminoglycan; IL-1β, interleukin-1β; NO, nitric oxide; W. som, Withania somnifera.

Figure 3 Gene expression of cartilage-modifying agents in herbal-treated chondrocytes.

Notes: Cells preexposed to IL-1β were treated with 100 μg/mL of herbals extracts. Gene expression of MMPs (A), inflammatory cytokines (B), anabolic agents (C), signaling pathways (D), and the activity of inhibitors on signaling pathways (E) were evaluated using quantitative real-time-polymerase chain reaction. Values correspond to means ± standard error from two independent experiments run in triplicate; *P<0.05; **P<0.01.
Abbreviations: ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; C. quad, Cissus quadrangularis; COX2, cyclooxygenase-2; DMSO, dimethyl sulfoxide; MMPs, matrix metalloproteinases; NOS2, nitric oxide synthase 2; W. som, Withania somnifera; IL-1β, interleukin-1β; MMP, matrix metal loproteinase; MAPK, mitogen-activated protein kinase; NF-κB, nuclear factor kappa B; TGF-β, transforming growth factor-β; TNF-α, tumour necrosis factor-α; IGF, insulin-like growth factor; PGE₂, prostaglandins 2; JNK, c-Jun N-terminal kinases; ERK, extracellular signal-regulated kinases.

Figure 4 Protein expression of cartilage-modifying agents in herbal-treated chondrocytes.

Notes: Cells preexposed to IL-1β were treated with 100 μg/mL of herbals extracts. Protein expression in chondrocytes was analyzed with fluorescence-activated cell sorting analysis. Values correspond to the mean ± standard error expression of survivin calculated from approximately 300 cells for each treatment.
Abbreviations: C. quadrangularis, Cissus quadrangularis; W. somnifera, Withania somnifera; IL-1β, interleukin-1β; MMP-3, matrix metalloproteinase-3; MAPK, mitogen-activated protein kinase.

Figure 5 Immunofluorescence analysis of survivin in chondrocytes.

Notes: OA chondrocytes were examined for the expression of survivin following treatment with herbals. Cells were stained with antigen-specific primary antibody followed by secondary antibody conjugated to tetramethylrhodamine dye. Nuclei were counterstained with DAPI. Cells were imaged using Leica confocal microscopy and processed in Photoshop. Representative images from two independent experiments are shown.
Abbreviations: IL-1β, interleukin-1β; OA, osteoarthritis.

Figure 6 Representative histopathological images of bone tissue and ALP activity in rats that underwent osteotomy.

Notes: (A) (a) Stained tissue sections of the control group, (b) Cissus quadrangularis-treated group, (c) Withania somnifera-treated group, and (d) combination group (original magnification ×10); (e) representative histopathological scores in osteotomized rats scaled from 0 to 11. Representative images from bone sections are presented. (B) Swiss albino rats surgically osteotomized and treated with herbal sample for 4 weeks were evaluated for levels of ALP in serum. (C) The structural differences of the osteotomized area of the limb were evaluated through X-ray and the observed severity of bone callus scaled between 0 and 5. (D) Representative radiographs showing the effects of different treatments; arrows indicate the area rats were osteotomized. Values correspond to means ± standard error (n=6 per group); *P<0.05.
Abbreviations: ALP, alkaline phosphatase; C. quad, Cissus quadrangularis; W. som, Withania somnifera.

Figure 7 Schematic diagram illustrating the therapeutic activity of Cissus quadrangularis.

Notes: C. quadrangularis inhibits catabolic activity of IL-1β-induced inflammation and cartilage damage. Reduction of proinflammatory cytokines and MMPs are regulated by treatment of C. quadrangularis.

Abbreviations: IL-1β, interleukin-1β; MAPK, mitogen-activated protein kinase; MMPs, matrix metalloproteinases; NO, nitric oxide; NF-κB, nuclear factor kappa B.