Liang-Ge-San: a classic traditional Chinese medicine formula, attenuates acute inflammation via targeting GSK3ß.

Sepsis is a serious life-threatening health disorder with high morbidity and mortality rates that burden the world, but there is still a lack of more effective and reliable drug treatment. Liang-Ge-San (LGS) has been shown to have anti-inflammatory effects and is a promising candidate for the treatment of sepsis. However, the anti-sepsis mechanism of LGS has still not been elucidated. In this study, a set of genes related to inflammatory chemotaxis pathways was downloaded from Encyclopedia of Genes and Genomes (KEGG) and integrated with sepsis patient information from the Gene Expression Omnibus (GEO) database to perform differential gene expression analysis. Glycogen synthase kinase-3ß (GSK-3ß) was found to be the feature gene after these important genes were examined using the three algorithms Random Forest, support vector machine recursive feature elimination (SVM-REF), and least absolute shrinkage and selection operator (LASSO), and then intersected with possible treatment targets of LGS found through the search. Upon evaluation, the receiver operating characteristic (ROC) curve of GSK-3ß indicated an important role in the pathogenesis of sepsis. Immune cell infiltration analysis suggested that GSK-3ß expression was associated with a variety of immune cells, including neutrophils and monocytes. Next, lipopolysaccharide (LPS)-induced zebrafish inflammation model and macrophage inflammation model was used to validate the mechanism of LGS. We found that LGS could protect zebrafish against a lethal challenge with LPS by down-regulating GSK-3ß mRNA expression in a dose-dependent manner, as indicated by a decreased neutrophils infiltration and reduction of inflammatory damage. The upregulated mRNA expression of GSK-3ß in LPS-induced stimulated RAW 264.7 cells also showed the same tendency of depression by LGS. Critically, LGS could induce M1 macrophage polarization to M2 through promoting GSK-3ß inactivation of phosphorylation. Taken together, we initially showed that anti-septic effects of LGS is related to the inhibition on GSK-3ß, both in vitro and in vivo.

Liang-Ge-San inhibits dengue virus serotype 2 infection by reducing caveolin1-induced cytoplasmic heat shock protein 70 translocation into the plasma membrane.

BACKGROUND: Dengue virus (DENV) is a major public health threat. However, there are no specific therapeutic drugs for DENV. Many Chinese heat-cleaning formulas, such as Liang-Ge-San (LGS), have been frequently used in the virus-induced diseases. The antiviral effect of LGS has not been reported yet. PURPOSE: In this study, the effect of LGS on the inhibition of dengue virus serotype 2 (DENV-2) was investigated and the relevant mechanism was explored. METHODS: High-performance liquid chromatography was applied to analyze the chemical characterization of LGS. The in vitro antiviral activities of LGS against DENV-2 were evaluated by time-of-drug-addition assay. The binding of heat shock protein 70 (Hsp70) and envelope (E) protein or caveolin1 (Cav1) were analyzed by immunofluorescence and immunoprecipitation assays. Then the role of Cav1 in the anti-DENV-2 effects of LGS was further examined. DENV-2 infected Institute of Cancer Research suckling mice (n = 10) and AG129 mice (n = 8) were used to examine the protective effects of LGS. RESULTS: It was found that geniposide, liquiritin, forsythenside A, forsythin, baicalin, baicalein, rhein, and emodin maybe the characteristic components of LGS. LGS inhibited the early stage of DENV-2 infection, decreased the expression levels of viral E and non-structural protein 1 (NS1) proteins. LGS also reduced E protein and Hsp70 binding and attenuated the translocation of Hsp70 from cytoplasm to the cell membrane. Moreover, LGS decreased the binding of Hsp70 to Cav1. Further study showed that the overexpression of Cav1 reversed LGS-mediated E protein and Hsp70 inhibition in the plasma membrane. In the in vivo study, LGS was highly effective in prolonging the survival time, reducing viral loads. CONCLUSION: This work demonstrates for the first time that LGS exerts anti-DENV-2 activity in vitro and in vivo. LGS decreases DENV-2-stimulated cytoplasmic Hsp70 translocation into the plasma membrane by Cav1 inhibition, thereby inhibiting the early stage of virus infection. These findings indicate that LGS may be a candidate for the treatment of DENV.

Optimal combination of anti-inflammatory components from Chinese medicinal formula Liang-Ge-San.

ETHNOPHARMACOLOGICAL RELEVANCE: Liang-Ge-San (LGS), a traditional Chinese medicine (TCM) formula, is usually used in acute inflammatory diseases in China. AIM OF THE STUDY: This study aims to detect the optimal combination of anti-inflammatory components from LGS. MATERIALS AND METHODS: Four mainly representative components (phillyrin, emodin, baicalin, and liquiritin) from LGS were chosen. The optimal combination was investigated by orthogonal design study. Zebrafish inflammation model was established by lipopolysaccharide (LPS)-yolk microinjection, and then the anti-inflammatory activities of different combinations were determined by survival analysis, changes on inflammatory cells infiltration, the MyD88/NF-κB and MAPK pathways and inflammatory cytokines production. RESULTS: The different combinations of bioactive ingredients from LGS significantly protected zebrafish from LPS-induced inflammation, as evidenced by decreased recruitment of macrophages and neutrophils, inhibition of the MyD88/NF-κB and MAPK pathways and down-regulation of TNF-α and IL-6. Among them, the combination group 8 most significantly protected against LPS. The combination of group 8 is: 0.1 µM of emodin, 2 µM of baicalin, 20 µM of phillyrin and 12.5 µM of liquiritin. CONCLUSION: The optimized combination group 8 exerts the most significant anti-inflammatory activity by inhibiting the recruitment of inflammatory cells, activation of the MyD88/NF-κB and MAPK pathways and the secretion of pro-inflammatory cytokines. This present study provides pharmacological evidences for the further development of new modern Chinese drug from LGS to treat acute inflammatory diseases, but indicated the use of zebrafish in the screening of components from formulas.

Liang-Ge-San, a classic traditional Chinese medicine formula, attenuates acute inflammation in zebrafish and RAW 264.7 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Liang-Ge-San (LGS) is a traditional Chinese medicine formula that commonly used in acute inflammatory diseases. However, the anti-inflammatory effects and the underlying mechanisms of LGS are not fully studied. AIM OF THE STUDY: This study aims to investigate the anti-inflammatory activity and explore the underlying mechanisms of LGS in zebrafish and cell inflammation models. MATERIALS AND METHODS: LPS-induced zebrafish inflammation model was established by LPS-yolk microinjection. The protective effect of LGS on zebrafish injected with LPS was observed using survival analysis. Infiltration of inflammatory cells was determined by H&E staining assay. Expression levels of key inflammatory cytokines TNF-α and IL-6 were measured by q-PCR assay. Recruitment of neutrophils and macrophages were observed by fluorescence microscopy, SB staining and NR staining. In vitro anti-inflammatory effects of LGS were evaluated on LPS-stimulated RAW 264.7 cells. The generation of IL-6 and TNF-α was detected by ELISA. The protein expression levels of JNK, p-JNK (Thr183/Tyr185), Nur77 and p-Nur77 (Ser351) were determined by Western blotting. Finally, two additional inflammatory models in zebrafish, which were induced by CuSO4 or tail fin injury, were also established and the recruitment of neutrophils and macrophages were observed for the determination of the anti-inflammatory activity of LGS. RESULTS: LGS protected zebrafish against LPS-induced death and dose-dependently inhibited LPS-induced acute inflammatory response in zebrafish, as indicated by increased survival rate, reduced infiltration of inflammatory cells, decreased recruitment of macrophages and neutrophils, and downregulated expression levels of TNF-α and IL-6. Additionally, LGS inhibited the secretion of TNF-α and IL-6, increased the expression of Nur77, and reduced the expression of p-Nur77 (Ser351) and p-JNK (Thr183/Tyr185) in LPS-stimulated RAW 264.7 cells. The anti-inflammatory action of LGS was also observed in another two zebrafish inflammation models, which was supported by the inhibition on neutrophils and macrophages recruitment. CONCLUSION: The present study demonstrates that LGS possesses anti-inflammatory activity in zebrafish inflammation models and LPS-stimulated RAW 264.7 cells, which is related to the inhibition on p-JNK and p-Nur77. This finding provides a pharmacological basis for LGS in the control of inflammatory disorder.

Liang-Ge-San, a Classic Traditional Chinese Medicine Formula, Attenuates Lipopolysaccharide-Induced Acute Lung Injury Through Up-Regulating miR-21.

Background: Acute lung injury (ALI) is a life-threatening disease without effective chemotherapy at present. Liang-Ge-San (LGS) is a famous traditional Chinese medicine formula, which is used to treat ALI in China. However, only a few studies have addressed the mechanisms of LGS in ALI. Purpose: To evaluate the anti-inflammatory effects of LGS on lipopolysaccharide (LPS)-induced ALI, and to explore its underlying molecular mechanism. Methods: Murine RAW264.7 cells were treated with LGS and LPS (1 µg/ml). The generation of IL-6, TNF-α, IL-1ß was detected by ELISA. The protein expressions of STAT3 and P-STAT3 (Tyr705) were determined by Western blotting and fluorescence confocal microscopy. STAT3 transcriptional activity was investigated by luciferase reporter gene assay. qPCR was used to detect the expressions of microRNA-21 (miR-21), STAT3, and IL-6. DSS cross-linking assay was used to assess the change of STAT3 dimer. In vivo anti-inflammatory effects of LGS were evaluated in an ALI mouse model induced by tracheal instillation of LPS (3 mg/kg). The anti-ALI effects were evaluated by ELISA, qPCR, Western blotting, BCA, and H&E assays. Results: LGS suppressed LPS-stimulated IL-6, TNF-α, and IL-1ß generation in murine macrophages RAW264.7. Moreover, LGS down-regulated protein levels of P-STAT3 (Tyr705) and STAT3, inhibited STAT3 transcriptional activity, and up-regulated miR-21. Furthermore, blockage of miR-21 antagonized the inhibitory effects of LGS on the production of IL-6 and the expressions of P-STAT3 (Tyr705) and STAT3 as well as the formation of STAT3 dimer. Critically, LGS up-regulated the expression of miR-21 and inhibited the protein expressions of STAT3 and P-STAT3 (Tyr705) to reduce the release of IL-6 and inflammatory cell infiltration as well as the degree of edema in LPS-induced ALI mice. Conclusion: LGS inhibited LPS-induced ALI through up-regulating miR-21 and subsequently inhibiting the STAT3 signaling pathway, thereby decreasing the release of IL-6.

Management of viral oral ulcers in children using Chinese herbal medicine: A report of two cases.

OBJECTIVE: Viral oral ulcers are common presentations in pediatric clinics. Although self-limiting, painful ulcerative lesions and inflamed mucosa can decrease oral intake and lead to dehydration. Despite the widespread use of Chinese herbal medicine (CHM) for pediatric upper respiratory disease in Taiwan, there is little evidence for its effectiveness as an antipyretic or in aiding ulcer healing for children with viral oral ulcers. We report two cases of children who presented with viral oral ulcers to illustrate the potential efficacy of CHM treatment in recovery from herpangina (HA) and herpetic gingivostomatitis (HGS). CLINICAL FEATURES AND OUTCOME: A 10-year-old girl with HA presented with an acute febrile illness associated with small vesicular or ulcerative lesions on the posterior oropharyngeal structures. The family refused western medicine due to a prior anaphylactic skin rash when she had taken sulfa drugs. The other patient was a 4-year-old boy with complaints of painful ulcers and hemorrhagic crusts on the lips. He was diagnosed with HGS and had received ibuprofen and supportive treatments such as hydration and local anesthesia spray for days, characterized by fever, anorexia, and nausea to no effect. Because the patients were suffering from the damp-heat syndrome according to Traditional Chinese Medicine (TCM) differentiation, both were treated using the same herbal formulas powder prescription, named Liang Ge San (LGS) and Gan Lu Xiao Du Dan (GLXDD). After several days of CHM treatment, the oral ulcers were in regression. Follow-up of the frontal view in both patients showed satisfactory disappearance of the sick furred tongue. CONCLUSIONS: The results of these case reports show that the early prescription of CHM is an effective modality of alternative treatment for viral oral ulcers. To our knowledge, this is the first report of CHM treatment hastening the recovery from febrile disease with viral oral ulcers in Taiwan. Future experimental studies to determine the definitive mechanism and clinical trials are warranted.

Liang-Ge-San, a classic traditional Chinese medicine formula, protects against lipopolysaccharide-induced inflammation through cholinergic anti-inflammatory pathway.

Liang-Ge-San (LGS) is a classic formula in traditional Chinese medicine, which is widely used to treat acute lung injury (ALI), pharyngitis and amygdalitis in clinic. However, the underlying mechanisms remain poorly defined. In this study, we discovered that LGS exerted potent anti-inflammatory effects in lipopolysaccharide (LPS)-induced inflammation. We found that LGS significantly depressed the production of IL-6 and TNF-α in LPS-stimulated RAW 264.7 macrophage cells. The degradation and phosphorylation of IκBα and the nuclear translocation of NF-κB p65 were also inhibited. Moreover, LGS activated α7 nicotinic cholinergic receptor (α7nAchR). The blockage of α7nAchR by selective inhibitor methyllycaconitine (MLA) or α7nAchR siRNA attenuated the inhibitory effects of LGS on IκBα, NF-κB p65, IL-6 and TNF-α. Critically, LGS significantly inhibited inflammation in LPS-induced ALI rats through the activation of NF-κB signaling pathway. However, these protective effects could be counteracted by the treatment of MLA. Taken together, we first demonstrated anti-inflammatory effects of LGS both in vitro and in vivo through cholinergic anti-inflammatory pathway. The study provides a rationale for the clinical application of LGS as an anti-inflammatory agent and supports the critical role of cholinergic anti-inflammatory pathway in inflammation.