Shikonin suppresses rheumatoid arthritis by inducing apoptosis and autophagy via modulation of the AMPK/mTOR/ULK-1 signaling pathway.

BACKGROUND: The overproliferation of fibroblast-like synoviocytes (FLS) contributes to synovial hyperplasia, a pivotal pathological feature of rheumatoid arthritis (RA). Shikonin (SKN), the active compound from Lithospermum erythrorhizon, exerts anti-RA effects by diverse means. However, further research is needed to confirm SKN's in vitro and in vivo anti-proliferative functions and reveal the underlying specific molecular mechanisms. PURPOSE: This study revealed SKN's anti-proliferative effects by inducing both apoptosis and autophagic cell death in RA FLS and adjuvant-induced arthritis (AIA) rat synovium, with involvement of regulating the AMPK/mTOR/ULK-1 pathway. METHODS: SKN's influences on RA FLS were assessed for proliferation, apoptosis, and autophagy with immunofluorescence staining (Ki67, LC3B, P62), EdU incorporation assay, staining assays of Hoechst, Annexin V-FITC/PI, and JC-1, transmission electron microscopy, mCherry-GFP-LC3B puncta assay, and western blot. In AIA rats, SKN's anti-arthritic effects were assessed, and its impacts on synovial proliferation, apoptosis, and autophagy were studied using Ki67 immunohistochemistry, TUNEL, and western blot. The involvement of AMPK/mTOR/ULK-1 pathway was examined via western blot. RESULTS: SKN suppressed RA FLS proliferation with reduced cell viability and decreased Ki67-positive and EdU-positive cells. SKN promoted RA FLS apoptosis, as evidenced by apoptotic nuclear fragmentation, increased Annexin V-FITC/PI-stained cells, reduced mitochondrial potential, elevated Bax/Bcl-2 ratio, and increased cleaved-caspase 3 and cleaved-PARP protein levels. SKN also enhanced RA FLS autophagy, featuring increased LC3B, reduced P62, autophagosome formation, and activated autophagic flux. Autophagy inhibition by 3-MA attenuated SKN's anti-proliferative roles, implying that SKN-induced autophagy contributes to cell death. In vivo, SKN mitigated the severity of rat AIA while also reducing Ki67 expression, inducing apoptosis, and enhancing autophagy within AIA rat synovium. Mechanistically, SKN modulated the AMPK/mTOR/ULK-1 pathway in RA FLS and AIA rat synovium, as shown by elevated P-AMPK and P-ULK-1 expression and decreased P-mTOR expression. This regulation was supported by the reversal of SKN's in vitro and in vivo effects upon co-administration with the AMPK inhibitor compound C. CONCLUSION: SKN exerted in vitro and in vivo anti-proliferative properties by inducing apoptosis and autophagic cell death via modulating the AMPK/mTOR/ULK-1 pathway. Our study revealed novel molecular mechanisms underlying SKN's anti-RA effects.

Therapeutic effects of shikonin on adjuvant-induced arthritis in rats and cellular inflammation, migration and invasion of rheumatoid fibroblast-like synoviocytes via blocking the activation of Wnt/ß-catenin pathway.

BACKGROUND: Shikonin (SKN), the main bioactive component isolated from Lithospermum erythrorhizon Sieb et Zucc, has multiple activities including anti-rheumatic effect, but its specific roles and the precise mechanisms in regulating biological properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) are unclear and need further clarification. PURPOSE: This study explored the therapeutic roles of SKN on rat adjuvant-induced arthritis (AIA) and cellular inflammation, migration and invasion of TNF-α-induced RA FLS (MH7A cells), and further demonstrated the involved mechanisms. METHODS: SKN was intraperitoneally given to AIA rats and its therapeutic role was valued. The effects of SKN in vivo and in vitro on the production of pro-inflammatory factors were examined by ELISA and western blot. Wound-healing, transwell and phalloidin staining assay were carried out to evaluate the effects of SKN on TNF-α-induced migration and invasion in RA FLS. The involvement of Wnt/ß-catenin pathway was checked by immunohistochemistry or immunofluorescence assay for ß-catenin and western blot for pathway-related proteins. RESULTS: SKN treatment in AIA rats reduced paw swelling, arthritis index and pathological damage of ankle joints, indicating its anti-arthritic effect in vivo. SKN had anti-inflammatory roles in vivo and in vitro, evidenced by inhibiting the production of pro-inflammatory factors (like IL-1ß, IL-6, IL-8, TNF-α, MMP-2 and MMP-9) in sera and synovium of AIA rats, and in TNF-α-induced MH7A cells. Gelatin zymography result revealed the suppression of SKN on TNF-α-induced MMP-2 activity in vitro. Moreover, SKN inhibited TNF-α-induced migration, invasion and cytoskeletal reorganization in MH7A cells. Mechanistically, SKN suppressed the activation of Wnt/ß-catenin signaling in AIA rat synovium and in TNF-α-induced MH7A cells, indicated by the reduced protein levels of Wnt1, p-GSK-3ß (Ser9) and ß-catenin, the raised protein level of GSK-3ß and the decreased nuclear translocation of ß-catenin. Interestingly, the combination of LiCl (Wnt/ß-catenin agonist) canceled the therapeutic functions of SKN on cellular inflammation, migration and invasion in TNF-α-induced MH7A cells, whereas XAV939 (Wnt/ß-catenin inhibitor) enhanced the therapeutic roles of SKN. CONCLUSION: SKN showed therapeutic effects on rat AIA and cellular inflammation, migration and invasion of TNF-α-stimulated RA FLS via interrupting Wnt/ß-catenin pathway.

Aucubin enhances the antitumor activity of cisplatin through the inhibition of PD-L1 expression in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a leading cause of cancer-associated mortality in the world. However, the anticancer effects of aucubin against HCC have yet to be reported. Cisplatin often decreased CD8+ tumor-infiltrating lymphocytes in the tumor microenvironment through increasing programmed death-ligand 1 (PD-L1) expression, which seriously affected the prognostic effect of cisplatin in the treatment of patients with HCC. Therefore, it is necessary to identify a novel therapeutic avenue to increase the sensitivity of cisplatin against HCC. PURPOSE: This study aims to evaluate the anti-tumor effect of aucubin on HCC, and also to reveal the synergistic effects and mechanism of aucubin and cisplatin against HCC. STUDY DESIGN AND METHODS: An H22 xenograft mouse model was established for the in vivo experiments. Cancer cell proliferation was detected by MTT assay. RT-qPCR was performed to analyze CD274 mRNA expression in vitro. Western blotting was employed to determine the expression levels of the PD-L1, p-Akt, Akt, p-ß-catenin, and ß-catenin in vitro. Immunofluorescence was carried out to examine ß-catenin nuclear accumulation in HCC cells. Immunohistochemistry was used to detect tumoral PD-L1 and CD8α expression in xenograft mouse model. RESULTS: Aucubin inhibits tumor growth in a xenograft HCC mouse model, but did not affect HCC cell viability in vitro. Aucubin treatment significantly inhibited PD-L1 expression through inactivating Akt/ß-catenin signaling pathway in HCC cells. Overexpression of PD-L1 dramatically reversed aucubin-mediated tumoral CD8+ T cell infiltration and alleviated the antitumor activity of aucubin in xenograft mouse model. Moreover, Cisplatin could induce the expression of PD-L1 through the activation of the Akt/ß-catenin signaling pathway in HCC cells, which can be blocked by aucubin in vitro. In xenograft mouse model, cisplatin treatment induced PD-L1 expression and alleviated the infiltration of CD8+ T lymphocytes in the tumor microenvironment. Aucubin not only abrogated cisplatin-induced PD-L1 expression but also enhanced the antitumor efficacy of cisplatin in a mouse xenograft model of HCC. CONCLUSION: Aucubin exerts antitumor activity against HCC and also enhances the antitumor activity of cisplatin by suppressing the Akt/ß-catenin/PD-L1 axis.

Umbelliferone Inhibits Migration, Invasion and Inflammation of Rheumatoid Arthritis Fibroblast-Like Synoviocytes and Relieves Adjuvant-Induced Arthritis in Rats by Blockade of Wnt/ß-Catenin Signaling Pathway.

Umbelliferone (UMB), a natural coumarin compound, has been reported to possess anti-rheumatic effects on rheumatoid arthritis (RA) experimental models, but its potential role of UMB in regulating migration, invasion and inflammation of RA fibroblast-like synoviocytes (FLS) remain unclear. Herein, MTT assay was performed to confirm the non-cytotoxic concentrations (10, 20, and 40[Formula: see text][Formula: see text]M) and the treatment time (24[Formula: see text]h) of UMB on TNF-[Formula: see text]-stimulated RA FLS (MH7A cells) in vitro. Results of wound-healing, transwell and phalloidin staining assays revealed that UMB inhibited TNF-[Formula: see text]-induced migration, invasion and F-actin cytoskeletal reorganization in MH7A. Results of ELISA, western blot and gelatin zymography indicated that UMB decreased the productions of pro-inflammatory factors, including IL-1[Formula: see text], IL-6, IL-8, MMP-2 and MMP-9, and inhibited MMP-2 activity in TNF-[Formula: see text]-stimulated MH7A cells. In vivo, UMB (25[Formula: see text]mg/kg and 50[Formula: see text]mg/kg) relieved the joint damage and synovial inflammation in rats with adjuvant-induced arthritis (AIA). Mechanistically, UMB could suppress Wnt/[Formula: see text]-catenin signaling both in TNF-[Formula: see text]-induced MH7A cells and in AIA rat synovium, evidenced by decreasing Wnt1 protein level, activating GSK-3[Formula: see text] kinase by blocking GSK-3[Formula: see text] (Ser9) phosphorylation, and reducing the protein level and nuclear translocation of [Formula: see text]-catenin. Importantly, combined use of lithium chloride (a Wnt/[Formula: see text]-catenin signaling agonist) eliminated the inhibitory effects of UMB on migration, invasion and inflammation in vitro and the anti-arthritic effects of UMB in vivo. We concluded that UMB inhibited TNF-[Formula: see text]-induced migration, invasion and inflammation of RA FLS and attenuated the severity of rat AIA through its ability to block Wnt/[Formula: see text]-catenin signaling pathway.

[Effect of wuhu decoction on surface co-stimulation molecule expression of peripheral dendritic cells in infants with asthma].

OBJECTIVE: To study the effect of Wuhu Decoction (WHD) on the expression of co-stimulation molecule of peripheral dendritic cells (DC), CD80, CD83 and CD86, in infants with asthma, in order to provide practical basis for further elucidate the action mechanism of WHD in preventing and treating infantile asthma. METHODS: Sixty infants with asthma of Fei phlegm-heat accumulation syndrome type were randomized into the treatment group treated with WHD and the control group treated with Western medicine (fluticasone propionate oral taking or inhalation). And 10 healthy infants were set as normal control. With Thomas method adopted, the DC were isolated from peripheral blood of all infants subjected. The expressions of surface co-stimulation molecules of DC, CD80, CD83 and CD86, were detected by flow cytometry. Their changes before and after treatment in different groups were analyzed and compared. RESULTS: Expressions of CD80 and CD86 of peripheral blood DC in asthmatic infants were remarkably higher than those in the normal control (P<0.01). In the treated group, CD80 expression lowered from 18.06 +/- 4.53 before treatment to 13.18 +/- 3.02 after treatment and CD86 expression lowered from 38.61 +/- 10.54 to 29.65 +/- 8.55; while in the control group, the two expressions were lowered from 18.40 +/- 3.86 to 15.34 +/- 3.90, and from 38.29 +/- 11.67 to 35.88 +/- 13.85 respectively, the lowering in both groups were statistically significant (P<0.01 and P<0.05), but it was more significant in the treated group (P<0.05). As for CD83, no significant difference existed between groups and no change was found in either group after treatment (P>0.05). CONCLUSION: WHD can regulate the co-stimulation molecules of dendritic cells in asthma infants to reduce the expressions of CD80 and CD86.

[Clinical study on relationship between sluggishness of lung-defensive qi and levels of vasoactive intestinal polypeptide and thromboxane B2].

OBJECTIVE: To explore the nature of pathology of sluggishness of lung-defensive qi and to offer objective experimental indexes for weifen syndrome (defensive phase syndrome). METHODS: According to the completely random design, the plasma levels of vasoactive intestinal peptide (VIP) and thromboxane B2 (TX2) of 19 patients with weifen syndrome and 13 patients with qifen syndrome (qi phase syndrome) were detected by radioimmunoassay. The plasma levels of VIP and TX2 at different stages of weifen syndrome and qifen syndrome were observed. RESULTS: The plasma levels of VIP in weifen syndrome and in the late stage of weifen syndrome increased greatly at different stages as compared to qifen syndrome and the blank group (P < 0.01), while the plasma level of TX2 of weifen syndrome was higher only at the late stage than the blank group and qifen syndrome (P < 0.01). As for the levels of VIP and TX2 in weifen syndrome with different internal organs infected, there was no significant difference (P > 0.05). CONCLUSION: VIP may be an index reflecting the pathology of weifen syndrome, and it is one of the material foundations of sluggishness of lung-defensive qi, but it has nothing to do with the infected internal organs. The level of TX2 increases only after the fever of patients with weifen syndrome subsided, so it can not be the basis for diagnosis of the early stage of weifen syndrome. It doesn't increase in qifen syndrome either, the mechanism remains to be further studied.

[Clinical study on effect of jiexiao oral liquid in preventing and curing virus induced asthma in children].

OBJECTIVE: To observe the effect and the possible mechanism of Jiexiao Oral Liquid (JXOL) in preventing and curing virus induced asthma in children. METHODS: One hundred and sixty patients of acute upper respiratory tract infection (AURTI) with asthma history were randomly divided into 2 groups. JXOL was given to the treated group within 24 hrs after occurrence of AURTI symptoms, and virazole of 10-15 mg.kg-1.d-1 was given to the control group, the therapeutic course for both groups was 7 days. Changes of clinical symptoms, signs, therapeutic effect, pulmonary function and immuno-globulin in patients were observed. RESULTS: The total effective rate of the treated group was 83.8%, the clinical control rate was 48.8%, while those in the control group were 62.5% and 23.8% respectively, showing significant difference between them (P < 0.01). JXOL could obviously improve the indexes of forced expiratory volume in one second (FEV1) and peak expiratory flow (PEF), reduce the level of IgE, as compared with those before treatment, the difference was significant (P < 0.01 or P < 0.05); it also showed significant difference as compared with those in the control group after treatment. The changes of IgA, IgG and IgM after treatment showed insignificant difference. CONCLUSION: JXOL was effective in preventing and curing virus induced asthma in children, it also shows pulmonary function improving and immune regulating effects.