Saikosaponin D alleviates inflammatory response of osteoarthritis and mediates autophagy via elevating microRNA-199-3p to target transcription Factor-4

Abstract

Objective: This study was to investigate the underlying mechanism by which Saikosaponin D (SSD) mitigates the inflammatory response associated with osteoarthritis (OA) and regulates autophagy through upregulation of microRNA (miR)-199-3p and downregulation of transcription Factor-4 (TCF4).

Methods: A mouse OA model was established. Mice were intragastrically administered with SSD (0, 5, 10 μmol/L) or injected with miR-199-3p antagomir into the knee. Then, pathological changes in cartilage tissues were observed. Normal chondrocytes and OA chondrocytes were isolated and identified. Chondrocytes were treated with SSD and/or transfected with oligonucleotides or plasmid vectors targeting miR-199-3p and TCF4. Cell viability, apoptosis, inflammation, and autophagy were assessed. miR-199-3p and TCF4 expressions were measured, and their targeting relationship was analyzed.

Results: In in vivo experiments, SSD ameliorated cartilage histopathological damage, decreased inflammatory factor content and promoted autophagy in OA mice. miR-199-3p expression was downregulated and TCF4 expression was upregulated in cartilage tissues of OA mice. miR-199-3p expression was upregulated and TCF4 expression was downregulated after SSD treatment. Downregulation of miR-199-3p attenuated the effect of SSD on OA mice. In in vitro experiments, SSD inhibited the inflammatory response and promoted autophagy in OA chondrocytes. Downregulation of miR-199-3p attenuated the effect of SSD on OA chondrocytes. In addition, upregulation of miR-199-3p alone inhibited inflammatory responses and promoted autophagy in OA chondrocytes. miR-199-3p targeted TCF4. Upregulation of TCF4 attenuated the effects of miR-199-3p upregulation on OA chondrocytes.

Conclusions: SSD alleviates inflammatory response and mediates autophagy in OA via elevating miR-199-3p to target TCF4.

Keywords: Autophagy; MicroRNA-42; Osteoarthritis; Saikosaponin D; Transcription Factor-4.

Fig. 1

SSD relieves cartilage damage in OA mice via elevating miR-199-3p. A HE staining observed articular cartilage damage. The results showed that AC tissue in Sham group showed smooth surface and complete structure; while, OA cartilage tissue showed rough surface and cartilage destruction. SSD treatment improved cartilage damage, and downregulation of miR-199-3p weakened the protective effect of SSD on OA cartilage, arrows indicate cartilage damage; B Safranine O-fast green staining observed AC damage and showed that chondrocytes were regularly arranged in Sham group; while, the cartilage tissue degradation of OA mice was severe. SSD treatment could reduce the degradation of cartilage tissue, and downregulation of miR-199-3p weakened the protective effect of SSD on OA cartilage, arrows indicate cartilage degradation; C OARSI score evaluated the injury of AC, and the results showed that OARSI score increased in OA mice. SSD treatment could reduce OARSI score, and downregulation of miR-199-3p decreased the effect of SSD; D–E TUNEL staining tests showed that apoptosis of articular chondrocytes increased in OA mice, arrows indicate chondrocyte apoptosis. SSD treatment reduced the apoptosis of articular chondrocytes, and down-regulating miR-199-3p weakened the inhibitory effect of SSD on the apoptosis of OA chondrocytes; F RT-qPCR detected mRNA expression of miR-199-3p and TCF4. The results showed that miR-199-3p was downregulated and TCF4 mRNA was upregulated in the cartilage tissue of OA mice. SSD treatment promoted miR-199-3p expression and inhibited TCF4 mRNA, and downregulation of miR-199-3p decreased the effect of SSD; G–H Western blot detected TCF4 protein expression. TCF4 was upregulated in the cartilage tissue of OA mice. SSD treatment inhibited TCF4 protein expression, and downregulation of miR-199-3p decreased the effect of SSD. A,B,D, scale bar = 20 μm. A–D, the mice in each group. * P < 0.05, ** P < 0.01. n = 10

Fig. 2

SSD relieves inflammation and mediates autophagy in OA mice via augmenting miR-199-3p. A ELISA measured content of pro-inflammatory cytokines IL-1β, TNF-α, and IL-6. The contents of IL-1β, TNF-α, and IL-6 in the cartilage tissues of OA mice were increased. SSD treatment reduced the contents of IL-1β, TNF-α and IL-6, and downregulation of miR-199-3p weakened the effect of SSD; B–C Western blot measured autophagy-associated protein Beclin1 and LC3-II/LC3-I ratio. Beclin1 and LC3-II/LC3-I ratio in OA mice cartilage decreased; while, SSD treatment increased Beclin1 and LC3-II/LC3-I ratio, and downregulation of miR-199-3p weakened the effect of SSD; D RT-qPCR detected autophagy-related genes (ATG1 and PI3K). ATG1 expression decreased and PI3K expression increased in OA mouse cartilage; while, SSD treatment increased ATG1 expression and decreased PI3K expression, and downregulation of miR-199-3p weakened the effect of SSD; E–F Immunohistochemical detected LC3-II protein showed that the positive expression of LC3-II decreased in the cartilage tissue of OA mice; while, SSD treatment increased the positive expression of LC3-II, arrow indicating positive expression of LC3-II. Downregulation of miR-199-3p weakened the effect of SSD. * P < 0.05, ** P < 0.01. n = 10

Fig. 3

Isolation and identification of chondrocytes. A Toluidine blue staining identified chondrocytes. The chondrocytes isolated from rats in Sham group and OA group were blue and purple; while, the chondrocytes in OA group were reduced in number, sparsely arranged and with light staining; B Type II collagen staining observed the morphology of chondrocytes. COL2 was positive in the chondrocytes of rats in the Sham group, with brown particles visible in the cytoplasm, and the nucleus was basically unstained; COL2 staining was weak and positive in the chondrocytes of rats in the Model group, with a few light yellow particles in the cytoplasm, and the nucleus was basically unstained

Fig. 4

SSD relieves OA chondrocyte inflammation and controls autophagy via elevating miR-199-3p. A MTT analyzed cell proliferation. OA chondrocyte proliferation decreased; while, SSD treatment promoted cell proliferation. Downregulation of miR-199-3p attenuated the promoting effect of SSD on chondrocyte proliferation; B Flow cytometry measured cell apoptosis. OA chondrocyte apoptosis increased; while, SSD treatment inhibited apoptosis. Downregulation of miR-199-3p weakened the inhibitory effect of SSD on chondrocyte apoptosis; C ELISA measured content of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 in the cell supernatant. The contents of IL-1β, TNF-α and IL-6 in the supernatant of OA chondrocytes were increased; while, the contents of IL-1β, TNF-α and IL-6 were decreased by SSD treatment. Downregulation of miR-199-3p weakened the effect of SSD; D–E Western blot detected autophagy-correlated Beclin1 and LC3-II/LC3-I ratio. Beclin1 and LC3-II/LC3-I ratio decreased in OA chondrocytes; while, SSD treatment increased Beclin1 and LC3-II/LC3-I ratio. Downregulation of miR-199-3p weakened the effect of SSD; F RT-qPCR evaluated miR-199-3p and TCF4 mRNA expression. The expression of miR-199-3p was downregulated and the expression of TCF4 mRNA was upregulated in OA chondrocytes. SSD treatment could promote the expression of miR-199-3p and inhibit the expression of TCF4 mRNA. Downregulation of miR-199-3p weakened the effect of SSD; G–H Western blot detected TCF4 protein expression. The expression of TCF4 protein was upregulated in OA chondrocytes; while, SSD treatment inhibited the expression of TCF4 protein. Downregulation of miR-199-3p weakened the effect of SSD. * P < 0.05, ** P < 0.01. N = 3

Fig. 5

MiR-199-3p alleviates inflammatory response of OA chondrocytes and stimulates autophagy via targeting TCF4. A Bioinformatics website predicted the binding site between miR-199-3p and TCF4; B The luciferase activity assay verified the targeting relationship between miR-199-3p and TCF4. The results show that Chondrocytes co-transfected with TCF4-WT and miR-199-3p mimic showed decreased luciferase activity; C–E RT-qPCR and Western blot detected TCF4 mRNA and protein expression. After upregulation of miR-199-3p, the expression of TCF4 mRNA and protein decreased; F MTT measured cell proliferation. Upregulation of miR-199-3p promoted chondrocyte proliferation. However, upregulation of TCF4 can weaken the promoting effect of upregulation of miR-199-3p on cell proliferation; G Flow cytometry detected cell apoptosis. Upregulation of miR-199-3p inhibited cell apoptosis. Upregulation of TCF4 can weaken the inhibitory effect of upregulation of miR-199-3p on apoptosis; H ELISA detected contents of pro-inflammatory cytokines IL-1β, TNF-α, IL-6 in the supernatant. Upregulation of miR-199-3p reduced the contents of IL-1β, TNF-α, and IL-6. Upregulation of TCF4 can reduce the effect of upregulation of miR-199-3p; I–J Western blot measured autophagy-correlated Beclin1 and LC3-II/LC3-I ratio. Upregulation of miR-199-3p reduced Beclin1 and LC3-II/LC3-I ratios. Upregulation of TCF4 can reduce the effect of upregulation of miR-199-3p. * P < 0.05, ** P < 0.01. N = 3