Eucalyptol, limonene and pinene enteric capsules attenuate airway inflammation and obstruction in lipopolysaccharide-induced chronic bronchitis rat model via TLR4 signaling inhibition.

BACKGROUND: Chronic bronchitis (CB), a type of chronic obstructive pulmonary disease (COPD), poses a significant global health burden owing to its high morbidity and mortality rates. Eucalyptol, limonene and pinene enteric capsules (ELPs) are clinically used as expectorants to treat various respiratory diseases, including CB, but their acting mechanisms remain unclear. In this study, we investigated the anti-CB effects of ELP in a rat model of lipopolysaccharide (LPS)-induced CB. The molecular mechanisms underlying its inhibitory effects on airway inflammation were further explored in LPS-stimulated Beas-2B cells. METHODS: ELP was characterized using gas chromatography. The production of inflammatory mediators in bronchoalveolar lavage fluid (BALF) was determined using an enzyme-linked immunosorbent assay. The expression of MUC5AC, MUC5B, and p-p65 in the lung tissue was measured using immunohistochemical staining. The gene expression of inflammatory mediators was determined using qRT-PCR. The expression levels of the target proteins were detected by western blotting. Nuclear localization of p65 was determined using an immunofluorescence assay. RESULTS: Compared to the CB model rats, ELP-treated rats showed reduced airway resistance, inflammation, and goblet cell hyperplasia. In BALF, ELP decreased the levels of inflammatory mediators, including TNF-α, IL-6, MIP-1α, and CCL5. ELP also suppressed LPS-induced elevation of MUC5AC, MUC5B, and p-p65 in the lung tissue. The metabolic pathway changes caused by LPS challenge were improved by ELP treatment. In LPS-exposed Beas-2B cells, ELP treatment inhibited the expression of TNFA, IL6, CCL5, MCP1, and MIP2A and decreased the phospho-levels of toll-like receptor 4 (TLR4) signaling-related proteins, including p-p38, p-JNK, p-ERK, p-TBK1, p-IKKα/ß, p-IκB, p-p65, and p-c-Jun. ELP also hindered the nuclear translocation of p65, c-Jun, and IRF3. CONCLUSIONS: This study showed that ELP has a potential therapeutic effect in LPS-induced CB rat model, possibly by suppressing TLR4 signaling. These results justify the clinical use of ELP for the treatment of pulmonary inflammatory diseases.

Xuelian injection ameliorates complete Freund's adjuvant-induced acute arthritis in rats via inhibiting TLR4 signaling.

Background: Xuelian injection (XI), a classic preparation extracted from Saussureae Involucratae Herba, has been clinically used to manage rheumatoid arthritis (RA) for nearly twenty years in China. However, the underlying anti-RA mechanism of XI remains unclear. In this study, complete Freund's adjuvant (CFA)-induced acute arthritic model was used to examine the anti-RA effects of XI in vivo. The molecular mechanisms of this action were further investigated using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods: XI and XI freeze dried powder were characterized by UPLC analysis. CD68 and TLR4 expression in the ankle joints was measured by immunohistochemistry. The secretion of inflammatory mediators was detected by ELISA. The expression levels of TLR4 involved components were measured by Western blotting. The localization of transcription factors was measured by immunofluorescence assay. Results: XI treatment ameliorated arthritic symptoms induced by CFA in the ankle joints of rats. The serum levels of inflammatory mediators, including TNF-α, MCP-1, and Rantes were decreased by XI treatment. The elevation of CD68 and TLR4 levels in ankle joints caused by CFA was suppressed by XI treatment. Moreover, XI treatment inhibited the secretion of nitric oxide and prostaglandin E2 in LPS-treated RAW264.7 macrophages. The expression of their enzymes iNOS and COX-2 was also decreased after XI treatment. The production of inflammatory mediators, including TNF-α, IL-6, IL-1ß, MCP-1, MIP-1α, and Rantes was reduced by XI treatment in LPS-stimulated RAW264.7 cells. The phosphorylation of p38, JNK, ERK, TBK1, IKKα/ß, IκB, p65, c-Jun, and IRF3 was reduced after XI treatment. Additionally, the expression levels of nuclear proteins of p65, c-Jun, and IRF3 were inhibited by XI treatment. Conclusions: Taken together, XI possesses potential anti-RA effect and the underlying mechanism may be closely associated with the inhibition of TLR4 signaling. Our findings provide further pharmacological justifications for the clinical use of XI in RA treatment.

Sichen Formula Ameliorates Lipopolysaccharide-Induced Acute Lung Injury via Blocking the TLR4 Signaling Pathways.

Purpose: Sichen (SC) formula is a classic prescription of Tibetan medicine. Due to its potential anti-inflammatory effect, the SC formula has been clinically used to treat respiratory diseases for many years in the Chinese Tibet region. The present study aimed to investigate the anti-inflammatory effect of SC and explore the underlying mechanisms. Methods: SC formula was characterized by HPLC analysis. The acute lung injury (ALI) mouse model was induced by direct intratracheal lipopolysaccharide (LPS) instillation, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. Meanwhile, RAW264.7 macrophages were stimulated by LPS. The contents of inflammatory mediators in the culture medium were determined by ELISA. Protein levels were determined by immunohistochemical staining or Western blotting. Nuclear localization of NF-κB, AP-1, and IRF3 was performed using immunofluorescence and Western blotting. Results: In the LPS-induced ALI mouse model, SC treatment suppressed the secretion of inflammatory mediators (TNF-α, IL-6, IL-1ß, MCP-1, MIP-1α, and RANTES) in BALF. SC treatment hindered the recruitment of macrophages. SC treatment also inhibited the expression of CD68, p-p65, and TLR4 in the lung tissue. In the LPS-exposed RAW264.7 cells, the cell viability was not changed up to 400 µg/mL of SC. SC concentration-dependently suppressed the production of nitric oxide, prostaglandin E2, TNF-α, IL-6, MCP-1, MIP-1α, and RANTES in LPS-challenged RAW264.7 cells. The expression levels of iNOS, COX-2, p-p38, p-JNK, p-ERK, p-TBK1, p-IKKα/ß, p-IκB, p-p65, p-c-Jun, and p-IRF3 were decreased after SC treatment. Moreover, the nuclear translocation of p65, c-Jun, and IRF3 was also blocked by SC treatment. Conclusion: SC treatment inhibited the inflammatory responses in LPS-induced ALI mouse model/RAW264.7 macrophages. The underlying mechanism of this action may be closely associated with the suppression of TLR4 signaling pathways. These research findings provide further pharmacological justifications for the medicinal use of SC in the management of respiratory diseases.

Schisandrin B mitigates hepatic steatosis and promotes fatty acid oxidation by inducing autophagy through AMPK/mTOR signaling pathway.

BACKGROUND: Schisandrin B (Sch B), which inhibits hepatic steatosis caused by non-alcoholic fatty liver disease (NAFLD), is one of the most active dibenzocyclooctadienes isolated from Schisandra chinensis (Turcz.) Baill with various pharmacological activities. In this study, the role of Sch B-induced autophagy in lipid-lowering activities of Sch B was examined and the underlying mechanisms were elucidated. METHODS: Free fatty acid (FFA)-stimulated HepG2 cells and mouse primary hepatocytes (MPHs) and high-fat diet (HFD)-fed mice were used as NAFLD models. The role of Sch B-induced autophagy in lipid-lowering effects of Sch B was assessed using ATG5/TFEB-deficient cells and 3-methyladenine (3-MA)-treated hepatocytes and mice. RESULTS: Sch B simultaneously active autophagy through AMPK/mTOR pathway and decreased the number of lipid droplets in FFA-treated HepG2 cells and MPHs. Additionally, siATG5/siTFEB transfection or 3-MA treatment mitigated Sch B-induced autophagy and activation of fatty acid oxidation (FAO) and ketogenesis in FFA-treated HepG2 cells and MPHs. Sch B markedly decreased hepatic lipid content and activated the autophagy through AMPK/mTOR pathway in HFD-fed mice. However, the activities of Sch B were suppressed upon 3-MA treatment. Sch B upregulated the expression of key enzymes involved in FAO and ketogenesis, which was mitigated upon 3-MA treatment. Moreover, changes in hepatic lipid components and amino acids may be related to the Sch B-induced autophagy pathway. CONCLUSION: These results suggested that Sch B inhibited hepatic steatosis and promoted FAO by activation of autophagy through AMPK/mTOR pathway. Our study provides novel insights into the hepatic lipophagic activity of Sch B and its potential application in the management of NAFLD.

Tibetan Medicine for Diabetes Mellitus: Overview of Pharmacological Perspectives.

Diabetes mellitus (DM) and its complications pose a major public health threat which is approaching epidemic proportions globally. Current drug options may not provide good efficacy and even cause serious adverse effects. Seeking safe and effective agents for DM treatment has been an area of intensive interest. As a healing system originating in Tibet, Traditional Tibetan Medicine (TTM) has been widely used by Tibetan people for the prevention and treatment of DM and its complications for hundreds of years. Tibetan Materia Medica (TMM) including the flower of Edgeworthia gardneri (Wall.) Meisn., Phyllanthi Fructus, Chebulae Fructus, Huidouba, and Berberidis Cortex are most frequently used and studied. These TMMs possess hypoglycemic, anti-insulin resistant, anti-glycation, lipid lowering, anti-inflammatory, and anti-oxidative effects. The underlying mechanisms of these actions may be related to their α-glucosidase inhibitory, insulin signaling promoting, PPARs-activating, gut microbiota modulation, islet ß cell-preserving, and TNF-α signaling suppressive properties. This review presents a comprehensive overview of the mode and mechanisms of action of various active constituents, extracts, preparations, and formulas from TMM. The dynamic beneficial effects of the products prepared from TMM for the management of DM and its complications are summarized. These TMMs are valuable materia medica which have the potential to be developed as safe and effective anti-DM agents.

Xiao Qing Long Tang essential oil exhibits inhibitory effects on the release of pro-inflammatory mediators by suppressing NF-κB, AP-1, and IRF3 signalling in the lipopolysaccharide-stimulated RAW264.7 cells.

Xiao Qing Long Tang (literally "Minor blue dragon decoction" in Chinese), a traditional Chinese formula, is prescribed to treat respiratory diseases. However, only few studies have been reported on its anti-inflammatory mechanisms. In this study, we investigated the inhibitory effects of Xiao Qing Long Tang essential oil on inflammatory mediators and explored the mechanisms of action of XQEO in the lipopolysaccharide (LPS)-stimulated RAW264.7 cells. XQEO was prepared via steam distillation and characterized by GC-MS analysis. MTT and Griess assays were used to measure cell viability and NO production, respectively. The mRNA expression and the production of LPS-induced pro-inflammatory cytokines (IL-1ß, IL-6, TNF-α, and IL-10) and chemokines (MCP-1, Rantes, and MIP-1α) were determined by real-time PCR and enzyme-linked immunosorbent assay, respectively. Furthermore, we determined the protein levels of the components of NF-κB, AP-1 and IRF3 signalling by Western blotting. Immunofluorescence assay was used to estimate the nuclear translocation of NF-κB, AP-1 and IRF3. The results showed that XQEO inhibited the secretion of NO and PGE2 and down-regulated the mRNA and protein levels of iNOS and COX-2. We also found that XQEO suppressed the LPS-induced overproduction of pro-inflammatory mediators. Moreover, XQEO inhibited the phosphorylation of NF-κB/p65, AP-1/c-Jun, and IRF3 by suppressing their upstream kinases, such as MAPKs, TBK1, Akt, IKKα/ß, and IκB, reducing the LPS-induced NF-κB, AP-1 and IRF3 translocation to the nucleus. These findings suggest that XQEO effectively suppresses the production of pro-inflammatory mediators possibly through the inhibition of NF-κB, AP-1, and IRF3 signalling in the LPS-stimulated RAW264.7 cells.

Edgeworthia gardneri (Wall.) Meisn. Water Extract Ameliorates Palmitate Induced Insulin Resistance by Regulating IRS1/GSK3ß/FoxO1 Signaling Pathway in Human HepG2 Hepatocytes.

The flower of Edgeworthia gardneri (Wall.) Meisn is commonly used in beverage products in Tibet and has potential health benefits for diabetes. However, the mechanisms underlying anti-insulin resistance (IR) action of the flower of E. gardneri are not fully understood. This study aims to investigate the effects of the water extract of the flower of E. gardneri (WEE) on IR in palmitate (PA)-exposed HepG2 hepatocytes. WEE was characterized by UPLC analysis. PA-treated HepG2 cells were selected as the IR cell model. The cell viability was determined using MTT assay. Moreover, the glucose consumption and production were measured by glucose oxidase method. The glucose uptake and glycogen content were determined by the 2-NBDG (2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose) glucose uptake assay and anthrone-sulfuric acid assay, respectively. The intracellular triglyceride content was detected by oxidative enzymic method. Protein levels were examined by Western blotting. Nuclear localization of FoxO1 was detected using immunofluorescence analyses and Western blotting. The expression of FoxO1 target genes was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The viability of PA-treated HepG2 cells was concentration-dependently increased by incubation with WEE for 24 h. WEE treatment remarkably increased the consumption and uptake of glucose in PA-exposed HepG2 cells. Moreover, treatment with WEE significantly decreased the PA-induced over-production of glucose in HepG2 cells. After exposure of HepG2 cells with PA and WEE, the glycogen content was significantly elevated. The phosphorylation and total levels of IRß, IRS1, and Akt were upregulated by WEE treatment in PA-exposed HepG2 cells. The phosphorylation of GSK3ß was elevated after WEE treatment in PA-treated cells. WEE treatment also concentration-dependently downregulated the phosphorylated CREB, ERK, c-Jun, p38 and JNK in PA-exposed HepG2 cells. Furthermore, the nuclear protein level and nuclear translocation of FoxO1 were also suppressed by WEE. Additionally, PA-induced changes of FoxO1 targeted genes were also attenuated by WEE treatment. The GLUT2 and GLUT4 translocation were also promoted by WEE treatment in PA-treated HepG2 cells. Taken together, WEE has potential anti-IR effect in PA-exposed HepG2 cells; the underlying mechanism of this action may be associated with the regulation of IRS1/GSK3ß/FoxO1 signaling pathway. This study provides a pharmacological basis for the application of WEE in the treatment of metabolic diseases such as type 2 diabetes mellitus.

Observation on therapeutic effect of electroacupuncture at Jiaji (EX-B 2) and points of bladder meridian mainly for lumbar disc herniation / 中国针灸

<p><b>OBJECTIVE</b>To compare the efficacy differences between electroacupuncture and medication in lumbar disc herniation.</p><p><b>METHODS</b>Eighty-five cases of lumbar disc herniation were randomly divided into an electroacupuncture group (45 cases) and a medication group (40 cases). The electroacupuncture group was treated with electroacupuncture at Jiaji (EX-B 2), Shenshu (BL 23), Qihaishu (BL 24), Guanyuanshu (BL 26), Dachangshu (BL 25) and Yangtlingquan (GB 34) etc., once a day; and the medication group was treated with oral administration of Fugui Gutong capsule (3 times a day, 4 capsule each time) and 0.3 g ibuprofen (once a day). The scores of clincial symptoms and therapeutic effect were observed before and after treatment. Results In the electroacupuncture group, the effective rate was 84.4% (38/45), which was superior to that of 65.0% (26/40) in the medication group (P < 0.05). After treatment, the scores of symptoms significantly decreased in the two groups (both P < 0.01), and the reduction of scores in electroacupuncture group was superior to that in medication group (P < 0.05, P < 0.01).</p><p><b>CONCLUSION</b>Electroacupuncture at Jiaji (EX-B 2) and points of Bladder Meridian mainly has a better therapeutic effect on lumbar disc herniation, which is superior to oral administration of fugui gutong capsule and ibuprofen.</p>