Inhibitory Effect Of Sinomenine Research Articles

Sinomenine Ameliorates Colitis-Associated Cancer by Modulating Lipid Metabolism via Enhancing CPT1A Expression.

Colitis-associated cancer (CAC), arising from long-lasting intestinal inflammation, is a common type of colorectal cancer. Sinomenine (SIN), the major active compound of Sinomenium acutum, displays excellent antitumor activity. In modern pharmacological research, SIN has been proved to arrest proliferation of human colon cancer cells in vitro, but its functional role and specific mechanism in CAC were still elusive. This study explored the molecular mechanism of SIN on CAC. The results showed that orally administered SIN could decrease the occurrence and development of CAC. Metabolomics results revealed SIN could reprogram metabolism in CAC mice by reversing 34 endogenous metabolites. Importantly, the most prominent metabolic alteration was lipid metabolism. Mechanistically, SIN improved lipid metabolism by enhancing the expression of CPT1A in CAC mice. Moreover, the inhibitory effect of SIN on the proliferation of human colon cancer cells was blunted via CPT1A inhibitor. The results of this study added further evidence of the molecular mechanisms that allow SIN to exert anti-CAC effect by facilitating lipid metabolism and reaffirmed its potential and distinctive role as a chemopreventive agent in CAC.

Suppression of macrophage migration by down-regulating Src/FAK/P130Cas activation contributed to the anti-inflammatory activity of sinomenine

A large number of macrophages in inflamed sites not only amplify the severity of inflammatory responses but also contribute to the deleterious progression of many chronic inflammatory diseases, autoimmune diseases and cancers. Macrophage migration is a prerequisite for their entry into inflammatory sites and their participation of macrophages in the pathologic processes. Inhibition of macrophage migration is therefore a potential anti-inflammatory mechanism. Moreover, alleviation of inflammation also prevents the macrophages infiltration. Sinomenine (SIN) is an alkaloid derived from the Chinese medicinal plant Sinomenium acutum. It has multiple pharmacological effects, including anti-inflammation, immunosuppression, and anti-arthritis. However, its anti-inflammatory molecular mechanisms and effect on macrophage migration are not fully understood. The purpose of this research was to investigate the pharmacological effects and the molecular mechanism of SIN on macrophage migration in vivo and in vitro as well as to elucidate its anti-inflammatory mechanisms associated with macrophage migration. Our results showed that SIN reduced the number of RAW264.7 cells migrating into inflammatory paws and blocked lipopolysaccharide (LPS)-induced RAW264.7 cells and bone marrow-derived macrophages (BMDMs) migration in vitro. Furthermore, SIN attenuated the 3D mesenchymal migration of BMDMs. The absence of macrophage migration after circulatory and periphery macrophages depletion led to a reduction in the severity of inflammatory response. In macrophages depleted (macrophages-/-) mice, as inflammatory severity decreased, RAW264.7 cells migration was suppressed. A non-obvious effect of SIN on the inflammatory response was found in macrophages-/- mice, while the inhibitory effect of SIN on RAW264.7 cells migration was still observed. Furthermore, the migration of RAW264.7 cells pre-treated with SIN was suppressed in normal mice. Finally, Src/focal adhesion kinase (FAK)/P130Cas axis activation, which supports macrophages mesenchymal migration, and iNOS expression, NO production, integrin αV and in integrin β3 expressions, which promote Src/FAK/P130Cas activation, were down-regulated by SIN. However, SIN had no obvious effect on the expression of the monocyte chemoattractant protein-1 (MCP-1), which is an important chemokine for macrophage migration. These results indicated that SIN significantly inhibited macrophage mesenchymal migration by down-regulating on Src/FAK/P130Cas axis activation. There was a mutual regulatory correlation between the inflammatory response and macrophage migration, and the effects of SIN on macrophage migration were involved in its anti-inflammatory activity.

Sinomenine Inhibited Interleukin-1β-Induced Matrix Metalloproteinases Levels via SOCS3 Up-Regulation in SW1353 Cells.

Matrix metalloproteinases (MMPs) are required for collagen degradation which play a key pathological role in arthritis progression. Herein, the effect of sinomenine (SN) on Interleukin 1 beta (IL-1β)-induced MMPs production and its underlying mechanism were explored in SW1353 cells. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that 200 and 400 µM SN significantly inhibited SW1353 cell proliferation, thus the lower dose of SN (25-100 µM) were used in the subsequent experiments. Notably, the increased mRNA and protein levels of suppressor of cytokine signaling (SOCS) 3 were dose-dependently induced by SN. SN significantly suppressed mRNA and protein levels of MMPs in IL-1β-induced SW1353 cells. Through Western blot analysis, SN showed inhibitory effect on IL-1β-induced TAK1 and p65 phosphorylation. Moreover, SN blocked the interaction of TRAF6 and TAK1 resulting in inactivation of IL-1β pathway. Mechanistically, the inhibitory effect of SN on MMPs levels alongside TRAF6 and TAK1 interactions was abrogated by silencing SOCS3. Moreover, SN did not inhibit TAK1 kinase activity. In TAK1 silencing cells, the levels of MMPs and p65 phosphorylation of SN-treatedcells were lower than dimethyl sulfoxide (DMSO)-treated cells, indicating that blocking interaction was not a unique way for SN to inhibit MMPs levels. Finally, SN significantly inhibited IL-6-induced Janus tyrosine kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) phosphorylation in SW1353 cells. The levels of JAK2 phosphorylation and MMPs did not show a significant difference between IL-6 + SOCS3-small interfering RNA (siRNA) + SN group and IL-6 + SOCS3-siRNA + DMSO group. These findings demonstrated that SOCS3 expression was increased by SN blocked IL-1β-induced interaction between TRAF6 and TAK1 as well as IL-6 pathway activation, thereby culminating in the inhibition of MMPs levels.

Inhibitory effect of sinomenine on lung cancer cells via negative regulation of α7 nicotinic acetylcholine receptor.

Lung cancer is the leading cause of cancer deaths worldwide, with a high morbidity and less than 20% survival rate. Therefore, new treatment strategies and drugs are needed to reduce the mortality of patients with lung cancer. α7 nicotinic acetylcholine receptor (α7 nAChR), as a receptor of nicotine and its metabolites, is a potential target for lung cancer treatment. Our previous studies revealed that sinomenine plays anti-inflammation roles via α7 nAChR and down-regulates the expression of this receptor, thus increasing the inflammatory response. Hence, sinomenine is possibly a natural ligand of this receptor. In the present study, the effects of sinomenine on lung cancer A549 cells and tumor-bearing mice were determined to investigate whether this alkaloid has an inhibitory effect on lung cancer via α7 nAChR. CCK-8 assay, wound-healing test, and flow cytometry were performed for cell proliferation, cell migration, and apoptosis analysis in vitro, respectively. Xenograft mice were used to evaluate the effects of sinomenine in vivo. Results showed that sinomenine decreased cell proliferation and migration abilities but increased the percentage of apoptotic cells. Tumor volume in tumor-bearing mice was significantly reduced after sinomenine treatment compared with that in the vehicle group mice (p<0.05). Furthermore, the effects of sinomenine were abolished by the α7 nAChR antagonist mecamylamine and the allosteric modulator PNU-120596, but no change occurred when the mice were pretreated with the muscarinic acetylcholine receptor antagonist atropine. Meanwhile, sinomenine suppressed α7 nAChR expression in vitro and in vivo, as well as the related signaling molecules pERK1/2 and ERK1/2 and the transcription factors TTF-1 and SP-1. By contrast, sinomenine up-regulated the expression of another transcription factor, Egr-1. These effects were restricted by mecamylamine and PNU but not by atropine. Results suggested that sinomenine can inhibit lung cancer via α7 nAChR in a negative feedback mode.

Inhibitory effect of sinomenine on growth and migration of human hypertrophic scar fibroblasts

Objective To study the inhibitory effect of sinomenine on the growth and migration of human hypertrophic scar fibroblasts. Methods The hypertrophic scars were removed and fibroblasts were cultured in 3 patients (mean age 34.04±6.36, shoulder and back in 2 patients and limbs in 1 patient). MTT assay was used to detect the effect of 25 μmol/L and 50 μmol/L of sinomenine on the proliferation of fibroblasts. Flow cytometry was used to analyze the cell cycle of fibroblasts treated with sinomenine. The apoptosis of fibroblasts treated with Sinomenine was observed by hechest 33258 staining. The effect of sinomenine on the migration of fibroblasts was examined by transwell assay. Western blotting was used to verify the regulatory effect of sinomenine on Ttransforming growth factor-β1 (TGF-β1), Cyclin D1, B cell lymphoma/leukemia-2 (bcl-2) and matrix metalloproteinase (MMP)-2. Results The inhibitory rates of sinomenine on fibroblasts were (13.14±5.51)% and (43.28±4.98)% respectively after 25 μmol/L and 50 μmol/L sinomenine treatment for 24 hours. The inhibitory effects of sinomenine on fibroblasts increased with the increase of drug concentration and the prolongation of action time. After sinomenine treatment, G1/S phase arrest was observed, the number of apoptotic cells increased and the number of cell migration decreased. Sinomenine can inhibit the expression of TGF-β1, Cyclin D1, bcl-2 and MMP-2 proteins, decreased respectively (0.84±0.01 vs. 0.16±0.04 vs. 0.17±0.03), (1.06±0.01 vs. 0.93±0.04 vs. 0.82±0.05), (0.55±0.03 vs. 0.34±0.01 vs. 0.28±0.01) and (0.64±0.01 vs. 0.28±0.04 vs. 0.14±0.02) significant difference between the two groups (t=11.040, 25.370; 18.880, 15.410; 15.760, 17.440; 19.600, 7.210, P<0.05). Conclusion Sinomenine can inhibit the growth and migration of human hypertrophic scar fibroblasts. Key words: Sinomenine; Hypertrophic scar; Fibroblasts; Proliferation; Apoptosis

Lipopolysaccharides-mediated injury to chondrogenic ATDC5 cells can be relieved by Sinomenine via downregulating microRNA-192.

Sinomenine (SIN) is an isoquinoline derived from Caulis Sinomenii that has been used to treat rheumatoid arthritis and osteoarthritis for several decades in China. This study aims to reveal the effects of SIN on mouse chondrogenic ATDC5 cells growth and inflammation. SIN was used to treat ATDC5 cells injured by lipopolysaccharides (LPS). The following parameters were determined for evaluating the treatment effects of SIN: cell viability, apoptosis, reactive oxygen species generation, and pro-inflammatory cytokines release. Besides, the expression of LPS-sensitive miRNA (miR-192) and the activation of NF-κB and MAPK signaling were studied to explain SIN's function. SIN with concentration of 30μM significantly attenuated LPS-induced cell damage via increasing cell viability, inhibiting apoptosis and reactive oxygen species generation, and declining IL-6 and TNF-α release. miR-192 was downregulated by SIN treatment. Restoration of miR-192 expression by miRNA transfection could significantly impede SIN's protective action. Besides, the inhibitory effects of SIN on the activation of NF-κB and MAPK signaling were attenuated by miR-192 overexpression. Furthermore, GDF11 was found to be a target gene of miR-192. LPS-mediated injury to chondrogenic ATDC5 cells can be relieved by SIN via downregulating miR-192 and subsequently repressing the activation of NF-κB and MAPK signaling.

Sinomenine inhibits the growth of glioma cells through STAT3 signal pathway

The present study was designed to examine effects of Sinomenine (SM) on glioma cells growth in vivo and in vitro. Cells growth and apoptosis were detected by MTT assay, TUNEL assay and flow cytometric analysis. In the study, SM treatment led to growth inhibition on a series of glioma cell lines, including U87, U373, U251, Hs683 and T98G. SM prevented U87 growth in the nude mice as well. Inhibitory effects of SM on U87 cells proliferation in vitro and in vivo were more effective than that of temozolomide (TMZ), and SM has synergistic effects with TMZ in the glioma therapy. SM induced apoptotic death in U87 cells via activation of caspase-3, caspase-8 and caspase-9, and down-regulation of HIAP, Bcl-2 and survivin. Moreover, we observed SM decreased the expression of phosphorylated STAT3 (p-STAT3) both in vivo and in vitro. Interestingly, using a specific activator of STAT3, we demonstrated overexpression of p-STAT3 impaired, SM mediated growth inhibition and apoptosis induction in the U87 cells. In summary, our results indicate SM induced growth suppression of human glioma cells through inhibiting phosphorylation of STAT3.

α7 Nicotinic Acetylcholine Receptor is a Novel Mediator of Sinomenine Anti-Inflammation Effect in Macrophages Stimulated by Lipopolysaccharide.

Sinomenine (SIN), an alkaloid derived from the plant Sinomenium acutum, has anti-inflammatory and analgesic effects and has been used for rheumatoid arthritis treatment in China. This study aims to verify the hypothesis that SIN acts on α7 nicotinic acetylcholine receptor (α7nAChR) to inhibit the activation of macrophages stimulated by lipopolysaccharide. The prototypical α7nAChR antagonist α-bungarotoxin and mecamylamine attenuated the effect of SIN on tumor necrosis factor-α and interleukin-6 in RAW264.7 murine macrophage-like cells and primary peritoneal macrophages of mouse induced by lipopolysaccharide. With the knockdown of α7nAChR expression in RAW264.7 cells by small interfering RNA, the inhibitory effect of SIN on tumor necrosis factor-α and interleukin-6 was reversed. Sinomenine decreased p65 expression in nuclear and increased IκBα expression in cytoplasm, and these effects were reversed by the α7nAChR small interfering RNA as well. These results indicate that the anti-inflammatory effects of SIN on macrophages in vitro depend on α7nAChR.

Sinomenine protects against ischaemic brain injury: involvement of co-inhibition of acid-sensing ion channel 1a and L-type calcium channels

Sinomenine (SN), a bioactive alkaloid, has been utilized clinically to treat rheumatoid arthritis in China. Our preliminary experiments indicated that it could protect PC12 cells from oxygen-glucose deprivation-reperfusion (OGD-R), we thus investigated the possible effects of SN on cerebral ischaemia and the related mechanism. Middle cerebral artery occlusion in rats was used as an animal model of ischaemic stroke in vivo. The mechanisms of the effects of SN were investigated in vitro using whole-cell patch-clamp recording, calcium imaging in PC12 cells and rat cortical neurons subjected to OGD-R. Pretreatment with SN (10 and 30 mg·kg(-1) , i.p.) significantly decreased brain infarction and the overactivation of calcium-mediated events in rats subjected to 2 h ischaemia followed by 24 h reperfusion. Extracellular application of SN inhibited the currents mediated by acid-sensing ion channel 1a and L-type voltage-gated calcium channels, in the rat cultured neurons, in a concentration-dependent manner. These inhibitory effects contribute to the neuroprotection of SN against OGD-R and extracellular acidosis-induced cytotoxicity. More importantly, administration of SN (30 mg·kg(-1) , i.p.) at 1 and 2 h after cerebral ischaemia also decreased brain infarction and improved functional recovery. SN exerts potent protective effects against ischaemic brain injury when administered before ischaemia or even after the injury. The inhibitory effects of SN on acid-sensing ion channel 1a and L-type calcium channels are involved in this neuroprotection.

Sinomenine reduces invasion and migration ability in fibroblast-like synoviocytes cells co-cultured with activated human monocytic THP-1 cells by inhibiting the expression of MMP-2, MMP-9, CD147

CD147 expressed by monocytes, macrophages, and synoviocytes cells can stimulate the production of matrix metalloproteinases (MMPs) associated with the development of rheumatoid arthritis (RA). We investigated the effects of Sinomenine (SIN) on invasion and migration ability and gene expression of CD147, MMP-2, MMP-9 of fibroblast-like synoviocytes cells (FLS) co-cultured with activated human monocytic THP-1 cells (A-THP-1) in vitro. SIN is a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum. FLS cells were co-cultured with THP-1 cells which were induced to differentiate into macrophages with phorbol 12-myristate 13-acetate (PMA). Cells were treated with different concentrations of SIN. Invasion and migration ability of cells was tested by transwell assays. Western blot analysis and zymographic analysis were adopted to detect the expression of CD147 and MMPs, respectively. RT-PCR was used to determine the expression of mRNA of CD147, MMP-2, and MMP-9. The invasion and migration ability of the co-cultured cells was significantly inhibited by SIN in a concentration-dependent fashion, and at the same time, the levels of CD147, MMP-2, MMP-9 were markedly down-regulated. This inhibitory effect was most notable at concentrations of 0.25 and 1.00 mM (P < 0.01). Our results point to a possible mechanism of SIN on treatment of RA is the inhibitory effect of SIN on cell invasion and migration ability, which strongly correlates with repressing the expression of CD147, MMP-2, and MMP-9.

The inhibitory effect of Sinomenine on immunological function in mice

AbstractSinomenine, an alkaloid isolated from the stem of Sinomenium acutum (Thunb.) Rehd. et Wils., is reported to have clinically good curative effects on arthritis and rheumatoid arthritis. Our investigation has shown that sinomenine inhibits both the humoral and cellular immunity of mice. The weights of spleen and thymus of mice treated with sinomenine (50–150 mg/kg/d, i.p.) were drastically reduced. The antisheep red blood cell antibody production and delayed‐type hypersensitivity reaction induced by sheep red blood cells were inhibited by sinomenine at doses of 50, 100 and 150 mg/kg/d; and the survival time of split‐heart transplants was also prolonged. In vitro, sinomenine suppressed the proliferation of splenic cells stimulated with LPS and ConA. These immunosuppressive actions of sinomenine afford a reasonable explanation for its clinical effects in the treatment of arthritis and rheumatoid arthritis.