Experimental evidence of shikonin as a novel intervention for anti-inflammatory effects.

Shikonin is a natural product with antioxidant and anti-inflammatory activities. The biological activity of shikonin is still not fully understood, as well as its association with innate immunity and immune and inflammatory bowel disease (IBD) in humans. In this study, the toxicity of shikonin on Raw264.7 cells was assayed by MTT, and polarization of inflammatory macrophages was determined by flow cytometry. The results showed that shikonin can inhibit the polarization of macrophages towards M1 type and significantly inhibited the production of NO in the concentration range of 0.5-1 µM. In addition, after treatment with shikonin, the production of IL-1ß and TNF-α was significantly decreased. After shikonin administration, the body weight loss and decrease of colon length were significantly suppressed in DSS-treated colitis C57BL/6 mice. The pro-inflammatory cytokines TNF-α and IL-1ß in colonic homogenate were significantly decreased. Shikonin treatment resulted in a notable improvement in the histopathological manifestations in DSS-treated animals at 25/50 mg/kg. Meanwhile, we found that shikonin can regulate differentiation of T helper 17 cell (Th17)/regulatory T cell (Treg), thereby regulating the balance of Th17/Treg cells and exerting an anti-inflammatory effect in IBD animal models. In conclusion, we found that shikonin protects against DSS-induced acute colitis by, among other things, reducing immune cell infiltration, polarizing macrophages, and regulating Th17/Treg differentiation, as well as by downregulating the release of inflammatory cytokines. These findings showed that shikonin can improve inflammation by affecting macrophage polarization. Our experimental data provide experimental evidence and theory basis for research on anti-inflammatory effects for the shikonin as health or functional food.

MiR-151b inhibits osteoblast differentiation via downregulating Msx2.

PURPOSE: MicroRNA-151b (miR-151b) showed altered expression in ovariectomized rat model of osteoporosis. This study established an ovariectomy-induced osteoporotic rat model to investigate the role of miR-151b in osteoblasts. METHODS: Eighteen female Sprague-Dawley (SD) rats were divided randomly into Sham and OVX group (n = 9). The transfections with different miRNAs and expression vectors were confirmed by RT-qPCR. The protein expression of Msx2 was detected by Western blots. The interaction between miR-151b and Msx2D was evaluated by RNA pull-down and dual luciferase reporter assay. RESULTS: The expression of miR-151b was significantly increased in femoral tissues of ovariectomy-induced osteoporotic rats. The expression of osteogenesis marker genes including RUNX2, ALP, OCN, OSX, and Msx2 were all significantly increased in osteogenic medium (OM) incubated primary osteoblasts and MC3T3-E1 cells. The interaction between miR-151b and Msx2 was confirmed by luciferase reporter assay and RNA pull-down. Moreover, overexpression of miR-151b significantly inhibited Msx2 in both MC3T3-E1 cells and primary osteoblasts, while miR-151b inhibitor had the opposite effect on the expression of Msx2. In addition, in primary osteoblasts and MC3T3-E1 cells, miR-151b overexpression, or Msx2 silence significantly decreased the expression of OSX, ALP, RUNX2, and OCN. CONCLUSION: MiR-151b could inhibit osteoblast proliferation, differentiation, and mineralization via downregulating Msx2 in both MC3T3-E1 cells and primary osteoblasts. MiR-151b might serve as a novel therapeutic target for osteoporosis. ABBREVIATIONS: miR-151b: microRNA-151b; miRNAs: microRNAs; Msx2: Msh homeobox 2; MAPK: mitogen-activated protein kinase; STAT: signal transducer and activator of transcription; SD: Sprague-Dawley; BMD: bone mineral density; qRT-PCR: quantitative reverse transcription PCR; MTT: methyl thiazolyl tetrazolium; OVX: ovariectomy; ALP: alkaline phosphatase.

circSKIL promotes the ossification of cervical posterior longitudinal ligament by activating the JNK/STAT3 pathway.

Ossification of the posterior longitudinal ligament (OPLL) is a hyperostotic spinal condition that involves genetic factors as well as non-genetic factors, and its underlying molecular mechanism is largely unknown. Recently, circular RNAs (circRNAs) have been attracting the attention of researchers since they have important regulatory roles in many diseases, including bone metabolism disorders. The present study aimed to investigate the role of circRNA SKI-like proto-oncogene (circSKIL) in OPLL disease progression. First, primary posterior longitudinal ligament cells from patients with cervical spondylotic myelopathy (CSM) without OPLL (control group) and CSM patients with OPLL (OPLL group) were isolated, and the expression levels of circSKIL in ligament cells was found to be significantly increased in the OPLL group compared with control. This result was also confirmed in OPLL tissues. Next, circSKIL was overexpressed in control ligament cells, and the proliferation, mineralization, and osteogenic differentiation of ligament cells were found to be significantly enhanced; the phosphorylation levels of both JNK and STAT3 were upregulated. By contrast, the knockdown of circSKIL in OPLL ligament cells inhibited proliferation, mineralization, and osteogenic differentiation and inactivated the JNK/STAT3 pathway. Therefore, circSKIL may have a significant role in osteogenic differentiation and could serve as a potential target to prevent OPLL progression.