Modified Si Miao Powder granules alleviates osteoarthritis progression by regulating M1/M2 polarization of macrophage through NF-κB signaling pathway

Abstract

Background: Osteoarthritis (OA) is a chronic degenerative disease mainly characterized by cartilage damage and synovial inflammation. Si Miao Powder, an herbal formula, was recorded in ancient Chinese medicine prescription with excellent anti-inflammatory properties. Based on the classical formula, the modified Si Miao Powder (MSMP) was developed with the addition of two commonly Chinese orthopedic herbs, which had the efficacy of strengthening the therapeutic effect for OA.

Methods: In the in vivo experiments, thirty-six 8-week-old male C57BL/6 mice were randomly divided into six groups: sham group, OA group, celecoxib group, low-MSMP group, middle-MSMP group, and high-MSMP group. OA mice were constructed by destabilization of medial meniscus (DMM) and treated with MSMP granules or celecoxib by gavage. The effects of MSMP on cartilage, synovitis and inflammatory factor of serum were tested. For in vitro experiments, control serum and MSMP-containing serum were prepared from twenty-five C57BL/6 mice. Macrophages (RAW264.7 cells) were induced by lipopolysaccharide (LPS) and then treated with MSMP-containing serum. The expression of inflammatory factors and the change of the NF-κB pathway were tested.

Results: In vivo, celecoxib and MSMP alleviated OA progression in the treated groups compared with OA group. The damage was partly recovered in cartilage, the synovial inflammatory were reduced in synovium, and the concentrations of IL-6 and TNF-α were reduced and the expression of IL-10 was increased in serum. The function of the middle MSMP was most effective for OA treatment. The results of in vitro experiments showed that compared with the LPS group, the MSMP-containing serum significantly reduced the expression levels of pro-inflammatory (M1-type) factors, such as CD86, iNOS, TNF-α and IL-6, and promoted the expression levels of anti-inflammatory (M2-type) factors, such as Arg1 and IL-10. The MSMP-containing serum further inhibited NF-κB signaling pathway after LPS induction.

Conclusion: The study demonstrated that MSMP alleviated OA progression in mice and MSMP-containing serum modulated macrophage M1/M2 phenotype by inhibiting the NF-κB signaling pathway. Our study provided experimental evidence and therapeutic targets of MSMP for OA treatment.

Keywords: NF-κB signaling pathway; inflammation; macrophage polarization; modified Si Miao Powder; osteoarthritis.

FIGURE 1

MSMP inhibited articular cartilage damage in OA mice. (A) HE staining of the articular cartilage tissues in mice. (B) Statistical analysis of the number of chondrocytes in each of the group. (C) Safranin O and fast green staining of the articular cartilage tissues in mice. (D) OARSI analysis in mice. Scale bar represents 100 μm (Date are mean ± SD, ^#### p < 0.0001, versus Sham group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus OA group).

FIGURE 2

MSMP affected cartilage matrix metabolism in OA mice. (A) Immunohistochemical staining of Col2a1. (B) Statistical analysis of the positive expression for Col2a1. (C) Immunohistochemical staining of Mmp13. (D) Statistical analysis of the positive expression for Mmp13. Scale bar represents 100 μm (Date are mean ± SD, ^#### p < 0.0001, versus Sham group; *p < 0.05, **p < 0.01, ****p < 0.0001, versus OA group).

FIGURE 3

MSMP inhibited synovial inflammation in OA mice. (A) HE staining of the synovium in mice. (B) Synovitis score in mice; Scale bar represents 100 μm (Date are mean ± SD, ^#### p < 0.0001, versus Sham group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus OA group).

FIGURE 4

MSMP was involved in immunoregulation of synovial macrophages. (A) Immunohistochemical staining of Arg1. (B) Statistical analysis of the positive expression for Arg1 in synovial tissue. (C) Immunohistochemical staining of iNOS. (D) Statistical analysis of the positive expression for iNOS in synovial tissue. Scale bar represents 100 μm (Date are mean ± SD, ^#### p < 0.0001, versus Sham group; **p < 0.01, ***p < 0.001, ****p < 0.0001, versus OA group).

FIGURE 5

MSMP affected the expression levels of serum IL-6, IL-10 and TNF-α in OA mice. (A) Expression level of serum IL-6; (B) Expression level of serum TNF-α; (C) Expression level of serum IL-10 (Date are mean ± SD, ^#### p < 0.0001, versus Sham group; **p < 0.01, ***p < 0.001, ****p < 0.0001, versus OA group).

FIGURE 6

MSMP-containing serum regulated M1/M2 polarization of macrophage. (A) Relative mRNA expressions of IL-6; (B) Relative mRNA expressions of TNF-α; (C) Relative mRNA expressions of iNOS; (D) Relative mRNA expressions of CD86; (E) Relative mRNA expressions of IL-10; (F) Relative mRNA expressions of Arg1 (Date are mean ± SD, ^#### P < 0.0001, versus Control group; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, versus LPS group).

FIGURE 7

MSMP-containing serum inhibited NF-κB pathway in macrophages after LPS induction. (A) Western blot analysis of p-p65, p65 and β-actin; (B) Statistical analysis of the Western blot results for p-p65/β-actin (%); (C) Statistical analysis of the Western blot results for p-p65/p65 (%); Quantification of the proteins was analyzed by the ImageJ software. (D) The immunofluorescence assay shows the blue fluorescence for DAPI, the red fluorescence for p-p65 and finally the merge plot; (E, F) The expression of p-p65 was analyzed by the ImageJ software. Scale bar represents 100 μm (Date are mean ± SD, ^#### p < 0.0001, versus Control group; ***p < 0.001, ****p < 0.0001, versus LPS group).

FIGURE 8

The schematic diagram shows experimental steps and therapeutic effects of MSMP on OA in vivo and in vitro. In the in vivo experiments, MSMP suppressed cartilage degeneration, synovial inflammation and affected the expression levels of inflammatory factors in the serum of OA mice; in the in vitro experiments, MSMP switched the phenotype of macrophages by inhibiting the NF-κB signalling pathway.