Exosomes derived from hepatitis B virus-infected hepatocytes promote liver fibrosis via miR-222/TFRC axis.

Exosomal miRNAs activates hepatic stellate cell (HSC) and promote fibrosis. miR-222 was found to be increased in hepatitis B virus (HBV)-infected hepatocytes, and ferroptosis was reported to ameliorate liver fibrosis (LF). Although miR-222 and ferroptosis have been implicated in LF, the association between miR-222 and ferroptosis and how they coordinate to regulate LF are still not explicit. This study investigates the roles of miR-222 and transferrin receptor (TFRC) in LF. Lipid reactive oxygen species (ROS) level was analyzed by flow cytometry. FerroOrange staining was used to measure intracellular iron level. Luciferase reporter assay was adopted to confirm the binding of miR-222 and TFRC. Real-time quantitative PCR and immunoblots were applied to analyze gene and protein expression. The results showed that supplementation of exosomes derived from HBV-infected LO2 cells remarkably enhanced LX-2 cell activation, evidenced by elevated hydroxyprolin (Hyp) secretion and α-SMA and COL1A2 expression. miR-222 was significantly increased in HBV-Exo. Overexpressing miR-222 upregulated cell viability, secretion of Hpy, and expression of α-SMA and COL1A2, which were all blocked by overexpression of TFRC. Further study showed that TFRC was a target of miR-222, and miR-222 promoted LX-2 cell activation through suppressing TFRC-induced ferroptosis in LX-2 cells. Exosomal miR-222 derived from HBV-infected hepatocytes promoted LF through inhibiting TFRC and TFRC-induced ferroptosis. This study emphasizes the significance of miR-222/TFRC axis in LF and suggests new insights in clinical decision making while treating LF. Exosomes derived from HBV-infected LO2 cells promote LX-2 cell activation and liver fibrosis in mouse Exosomal miR-222 derived from HBV-infected LO2 cells promotes LX-2 cell activation TFRC is a target of miR-222 and inhibits LX-2 cell activation induced by miR-222 miR-222 promotes LX-2 cell activation through inhibiting TFRC-induced ferroptosis.

Anti-inflammatory activity of Anchusa italica Retz. in LPS-stimulated RAW264.7 cells mediated by the Nrf2/HO-1, MAPK and NF-κB signaling pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Anchusa italica Retz. (Boraginaceae) is an important medicinal plant for the treatment of meningitis and pneumonia in traditional Uygur medicines. AIM OF THE STUDY: To clarify the anti-inflammatory activity of A. italica, to reveal its molecular mechanisms, and to discover the anti-inflammatory active ingredients. MATERIALS AND METHODS: Dried and crushed aerial parts of A. italica were extracted with 75% ethanol to yield crude extract (AICE) and AICE was fractionated to obtain petroleum ether extract (AIPE), dichloromethane extract (AIDE), ethyl acetate extract (AIEE), n-butanol extract (AIBE) and residues (AIW). By measuring the effects of AIPE, AIDE, AIEE, AIBE and AIW on cell viability and nitric oxide (NO) in Lipopolysaccharide (LPS) stimulated RAW264.7 cell lines, AIDE with the lowest cytotoxicity and NO contents was finally selected for further chemical and anti-inflammatory investigations. LC-MS/MS experiment was applied to analyze the chemical composition of AIDE. MTT and Griess methods were used to detect the cell viability and to quantify the nitrite levels in culture supernatants, respectively. Prostaglandin E2 (PGE2), interleukin-6 (IL-6), interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) production was examined by ELISA assays. Real-time quantitative PCR was used to detect the expression of hemeoxygenase-1 (HO-1), Nrf2-mediated quinone oxidoreductase 1 (NQO-1), glutathione S-transferase A 1 (GSTA1) and glutathione S-transferase M 1 (GSTM1) mRNA. Western blot analysis was employed to examine the protein expression and enzymatic activities. RESULTS: In preliminary anti-inflammatory screening, AIDE showed the lowest cytotoxicity and the most significant inhibitory effect on the production of NO (the inhibitory is 89%) induced by LPS among the tested five extracts. Thirty-three compounds including twenty-five triterpenoids were identified by LC-MS/MS analysis. AIDE could inhibit LPS-induced the over-expression of NO, IL-6, PGE2, IL-1ß and TNF-α and down-regulate the levels of extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), P38-MAPK (P38) and nuclear transcription factors κB-P65 (P65) phosphorylation. It promoted the mRNA expression level of HO-1, NQO-1, GSTA1 and GSTM1 and the protein expression level of nuclear factor erythroid 2-related factor 2 (Nrf2) and HO-1. After the treatment of AIDE, P65 nuclear translocation was inhibited and Nrf2 nuclear translocation was increased. In addition, the protein expression of pyrolytic relevant protein nod-like receptor family pyrin domain-containing 3 (NLRP3) and IL-1ß were decreased after the AIDE treatment. CONCLUSIONS: Anchusa italica Retz. exerted its anti-inflammatory activity by inhibiting the mitogen-activated protein kinase (MAPK), nuclear transcription factors κB (NF-κB) and pyrolytic relevant proteins, down-regulating inflammatory factor levels, and activating the Nrf2/HO-1 pathway. Triterpenoids might be its major active anti-inflammatory ingredients.

NMDA receptors and L-arginine/nitric oxide/cyclic guanosine monophosphate pathway contribute to the antidepressant-like effect of Yueju pill in mice.

The present study aims to evaluate the involvement of N-methyl-d-aspartate receptor and nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) system in antidepressant-like effects of Yueju pill (YJ), a Chinese herbal medicine. The immobility time in tail suspension test (TST) and forced swim test (FST) was used to assess the antidepressant effects. Prior administration of L-arginine (750 mg/kg, intraperitoneal [i.p.]), a NO synthase substrate that enhances NO signaling or sildenafil (5 mg/kg, i.p.), a phosphodiesterase 5 inhibitor that enhances cGMP, blunted the antidepressant-like activity of YJ (2.7 g/kg, i.g.). Co-treatment of ineffective dose of YJ (1.35 g/kg, i.g.) with one of the reagents that suppress the NO/cGMP signaling, including methylene blue (10 mg/kg, i.p.), an inhibitor of NO synthase; 7-NI (7-nitroinidazole, 30 mg/kg, i.p.), an nNOS specific inhibitor; L-NAME (10 mg/kg, i.p.), a non-specific inhibitor of NO synthase; and MK-801 (0.05 mg/kg, i.p.), an NMDA receptor antagonist, reduced the immobility time in TST and FST, compared with those in vehicle or single drug treatment groups. Neither above drugs alone or co-administrated with YJ affected locomotor activity or anxiety behavior in open field test. Thus, our results suggest that the antidepressant-like action of YJ may depend on the inhibition of NMDA/NO/cGMP pathway.

Xingshentongqiao decoction mediates proliferation, apoptosis, orexin-A receptor and orexin-B receptor messenger ribonucleic acid expression and represses mitogen-activated protein kinase signaling.

BACKGROUND: Hypocretin (HCRT) signaling plays an important role in the pathogenesis of narcolepsy and can be significantly influenced by Chinese herbal therapy. Our previous study showed that xingshentongqiao decoction (XSTQ) is clinically effective for the treatment of narcolepsy. To determine whether XSTQ improves narcolepsy by modulating HCRT signaling, we investigated its effects on SH-SY5Y cell proliferation, apoptosis, and HCRT receptor 1/2 (orexin receptor 1 [OX1R] and orexin receptor 2 [OX2R]) expression. The signaling pathways involved in these processes were also assessed. METHODS: The effects of XSTQ on proliferation and apoptosis in SH-SY5Y cells were assessed using cell counting kit-8 and annexin V-fluorescein isothiocyanate assays. OX1R and OX2R expression was assessed by quantitative real-time polymerase chain reaction analysis. Western blotting for mitogen-activated protein kinase (MAPK) pathway activation was performed to further assess the signaling mechanism of XSTQ. RESULTS: XSTQ reduced the proliferation and induced apoptosis of SH-SY5Y cells. This effect was accompanied by the upregulation of OX1R and OX2R expression and the reduced phosphorylation of extracellular signal-regulated kinase (Erk) 1/2, p38 MAPK and c-Jun N-terminal kinase (JNK). CONCLUSIONS: XSTQ inhibits proliferation and induces apoptosis in SH-SY5Y cells. XSTQ also promotes OX1R and OX2R expression. These effects are associated with the repression of the Erk1/2, p38 MAPK, and JNK signaling pathways. These results define a molecular mechanism for XSTQ in regulating HCRT and MAPK activation, which may explain its ability to treat narcolepsy.