RESUMO
This study aims to investigate the effect and mechanism of Maxingshigan Decoction on inflammation in the rat model of cough variant asthma(CVA). The SPF-grade SD rats of 6-8 weeks were randomized into normal, model, Montelukast sodium, and low-, medium-, and high-dose Maxing Shigan Decoction groups, with 8 rats in each group. The CVA rat model was induced by ovalbumin(OVA) and aluminum hydroxide sensitization and ovalbumin stimulation. The normal group and model group were administrated with equal volume of normal saline by gavage, and other groups with corresponding drugs by gavage. After the experiment, the number of white blood cells in blood and the levels of interleukin-6(IL-6), interleukin-10(IL-10), and tumor necrosis factor-α(TNF-α) in the serum were measured. The lung tissue was stained with hematoxylin-eosin(HE). Western blot was employed to determine the protein levels of nuclear factor-κB(NF-κB), Toll-like receptor 4(TLR4), myeloid differentiation protein(MyD88), and mitogen-activated protein kinase(MAPK) in the lung tissue. Real-time PCR was carried out to measure the mRNA levels of TLR4 and MyD88 in the lung tissue. Compared with the normal group, the model group showed increased white blood cells, elevated IL-6 and TNF-α levels(P<0.01), lowered IL-10 level(P<0.01), up-regulated protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK(P<0.01) and mRNA levels of TLR4 and MyD88(P<0.01) in the lung tissue. HE staining showed obvious infiltration of inflammatory cells around the airway and cell disarrangement in the model group. Compared with the model group, Montelukast sodium and high-dose Maxing Shigan Decoction reduced the white blood cells, lowered the IL-6 and TNF-α levels(P<0.01), and elevated the IL-10 level(P<0.01). Moreover, they down-regulated the protein levels of TLR4, MyD88, p-p65/NF-κB p65, p-p38 MAPK/p38 MAPK in the lung tissue(P<0.01) and the mRNA levels of TLR4 and MyD88 in the lung tissue(P<0.01). HE staining showed that Montelukast sodium and high-dose Maxing Shigan Decoction reduced inflammatory cell infiltration and cell disarrangement. The number of white blood cells, the levels of IL-10 and TNF-α in the serum, the protein levels of TLR4, MyD88, p-p65/NF-κB p65, and p-p38 MAPK/p38 MAPK, and the mRNA levels of TLR4 and MyD88 in the lung tissue showed no significant differences between the Montelukast sodium group and high-dose Maxing Shigan Decoction group. Maxing Shigan Decoction can inhibit airway inflammation in CVA rats by inhibiting the activation of TLR4/MyD88/NF-κB and p38 MAPK signaling pathways.
Assuntos
Acetatos , Variante Tussígena da Asma , Ciclopropanos , NF-kappa B , Quinolinas , Sulfetos , Ratos , Animais , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/metabolismo , Interleucina-10/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-6/genética , Interleucina-6/metabolismo , Ratos Sprague-Dawley , Ovalbumina , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Inflamação , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , RNA MensageiroRESUMO
To investigate the effect of Huazhi Rougan Granules(HZRG) on autophagy in a steatotic hepatocyte model of free fatty acid(FFA)-induced nonalcoholic fatty liver disease(NAFLD) and explore the possible mechanism. FFA solution prepared by mixing palmitic acid(PA) and oleic acid(OA) at the ratio of 1â¶2 was used to induce hepatic steatosis in L02 cells after 24 h treatment, and an in vitro NAFLD cell model was established. After termination of incubation, cell counting kit-8(CCK-8) assay was performed to detect the cell viability; Oil red O staining was employed to detect the intracellular lipid accumulation; enzyme-linked immunosorbnent assay(ELISA) was performed to measure the level of triglyceride(TG); to monitor autophagy in L02 cells, transmission electron microscopy(TEM) was used to observe the autophagosomes; LysoBrite Red was used to detect the pH change in lysosome; transfection with mRFP-GFP-LC3 adenovirus was conducted to observe the autophagic flux; Western blot was performed to determine the expression of autophagy marker LC3B-â /LC3B-â ¡, autophagy substrate p62 and silent information regulator 1(SIRT1)/adenosine 5'-monophosphate(AMP)-activated protein kinase(AMPK) signaling pathway. NAFLD cell model was successfully induced by FFA at 0.2 mmol·L~(-1) PA and 0.4 mmol·L~(-1) OA. HZRG reduced the TG level(P<0.05, P<0.01) and the lipid accumulation of FFA-induced L02 cells, while elevated the number of autophagosomes and autophagolysosomes to generate autophagic flux. It also affected the functions of lysosomes by regulating their pH. Additionally, HZRG up-regulated the expression of LC3B-â ¡/LC3B-â , SIRT1, p-AMPK and phospho-protein kinase A(p-PKA)(P<0.05, P<0.01), while down-regulated the expression of p62(P<0.01). Furthermore, 3-methyladenine(3-MA) or chloroquine(CQ) treatment obviously inhibited the above effects of HZRG. HZRG prevented FFA-induced steatosis in L02 cells, and its mechanism might be related to promoting autophagy and regulating SIRT1/AMPK signaling pathway.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Humanos , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Sirtuína 1/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Autofagia , FígadoRESUMO
This study aims to predict the material basis and mechanism of Dachengqi Decoction in the treatment of sepsis based on network pharmacology. The chemical constituents and targets of Dachengqi Decoction were retrieved from TCMSP, UniPot and DrugBank and the targets for the treatment of sepsis from OMIM and GeneCards. The potential targets of Dachengqi Decoction for the treatment of sepsis were screened by OmicShare. STRING database and Cytoscape 3.7.2 were used to construct the Chinese medicinal-active component-target-disease, active component-key target-key pathway, and protein-protein interaction(PPT) networks. The gene ontology(GO) term enrichment analysis and Kyoto encyclopedia of genes and genomes(KEGG) pathway enrichment analysis were performed by DAVID(P<0.05). Finally, the animal experiment was conducted to verify some targets and pathways. A total of 40 active components and 157 targets of the Dachengqi Decoction, 2 407 targets for the treatment of sepsis, and 91 common targets of the prescription and the disease were also obtained. The key targets were prostaglandin G/H synthase 2(PTGS2), prostaglandin G/H synthase 1(PTGS1), protein kinase cAMP-dependent catalytic-α(PRKACA), coagulation factor 2 receptor(F2 R), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic gamma subunit(PIK3 CG), dipeptidyl peptidase 4(DPP4), etc. A total of 533 terms and 125 pathways were obtained for the 91 targets. The main terms were the response to drug, negative regulation of apoptotic process, positive regulation of nitric oxide biosynthetic process and lipopolysaccharide-mediated signaling pathway, and the pathways included pathways in cancer, hepatitis B, and phosphatidylinositol 3-kinase and protein kinase B(PI3 K/Akt) signaling pathway. The animal experiment confirmed that Dachengqi Decoction can down-regulate inflammatory cytokines interleukin-1ß(IL-1ß), IL-6 and tumor necrosis factor α(TNF-α)(P<0.01). It could also reduce the wet/dry weight ratio of lung tissue, the level of myeloperoxidase(MPO) and the phosphorylation of PI3 K and Akt(P<0.01). These results indicated that Dchengqi Decoction could act on inflammation-related targets and improve sepsis by inhibiting PI3 K/Akt signaling pathway. The animal experiment supported the predictions of network pharmacology. Dachengqi Decoction intervenes sepsis via multiple components, multiple targets, and multiple pathways. The result lays a foundation for further research on the mechanism of Dachengqi Decoction in the treatment of sepsis.