This study aimed to parallelly investigate the cardioprotective activity of Cinnamomi Ramulus formula granules(CRFG) and Cinnamomi Cortex formula granules(CCFG) against acute myocardial ischemia/reperfusion injury(MI/RI) and the underlying mechanism based on the efficacy of "warming and coordinating the heart Yang". Ninety male SD rats were randomly divided into a sham group, a model group, CRFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, and CCFG low and high-dose(0.5 and 1.0 g·kg~(-1)) groups, with 15 rats in each group. The sham group and the model group were given equal volumes of normal saline by gavage. Before modeling, the drug was given by gavage once a day for 7 consecutive days. One hour after the last administration, the MI/RI rat model was established by ligating the left anterior descending artery(LAD) for 30 min ischemia followed by 2 h reperfusion except the sham group. The sham group underwent the same procedures without LAD ligation. Heart function, cardiac infarct size, cardiac patho-logy, cardiomyocyte apoptosis, cardiac injury enzymes, and inflammatory cytokines were determined to assess the protective effects of CRFG and CCFG against MI/RI. The gene expression levels of nucleotide-binding oligomerization domain-like receptor family pyrin domain protein 3(NLRP3) inflammasome, apoptosis-associated speck-like protein containing a CARD(ASC), cysteinyl aspartate specific proteinase-1(caspase-1), Gasdermin-D(GSDMD), interleukin-1β(IL-1β), and interleukin-18(IL-18) were determined by real-time quantitative polymerase chain reaction(RT-PCR). The protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD were determined by Western blot. The results showed that both CRFG and CCFG pretreatments significantly improved cardiac function, decreased the cardiac infarct size, inhibited cardiomyocyte apoptosis, and reduced the content of lactic dehydrogenase(LDH), creatine kinase MB isoenzyme(CK-MB), aspartate transaminase(AST), and cardiac troponin Ⅰ(cTnⅠ). In addition, CRFG and CCFG pretreatments significantly decreased the levels of IL-1β, IL-6, and tumor necrosis factor-α(TNF-α) in serum. RT-PCR results showed that CRFG and CCFG pretreatment down-regulated the mRNA expression levels of NLRP3, caspase-1, ASC, and downstream pyroptosis-related effector substances including GSDMD, IL-18, and IL-1β in cardiac tissues. Western blot revealed that CRFG and CCFG pretreatments significantly decreased the protein expression levels of NLRP3, caspase-1, GSDMD, and N-GSDMD in cardiac tissues. In conclusion, CRFG and CCFG pretreatments have obvious cardioprotective effects on MI/RI in rats, and the under-lying mechanism may be related to the inhibition of NLRP3/caspase-1/GSDMD signaling pathway to reduce the cardiac inflammatory response.
Male ; Animals ; Rats ; Rats, Sprague-Dawley ; Interleukin-18 ; Myocardial Reperfusion Injury ; NLR Family, Pyrin Domain-Containing 3 Protein ; Tumor Necrosis Factor-alpha ; Myocardial Infarction ; Caspase 1

This study aims to investigate the effect of Astragali Radix-Curcumae Rhizoma(AC) combination on the proliferation, migration, and invasion of colon cancer HT-29 cells based on epithelial-mesenchymal transition(EMT). HT-29 cells were respectively treated with 0, 3, 6 and 12 g·kg~(-1) AC-containing serum for 48 h. The survival and growth of cells were measured by thiazole blue(MTT) colorimetry, and the proliferation, migration, and invasion of cells were detected by 5-ethynyl-2'-deoxyuridine(EdU) test and Transwell assay. Cell apoptosis was examined by flow cytometry. The BALB/c nude mouse model of subcutaneous colon cancer xenograft was established, and then model mice were classified into blank control group, 6 g·kg~(-1) AC group, and 12 g·kg~(-1) AC group. The tumor weight and volume of mice were recorded, and the histopathological morphology of the tumor was observed based on hematoxylin-eosin(HE) staining. The expression of apoptosis-associated proteins B-cell lymphoma-2-associated X protein(Bax), cysteine-aspartic acid protease-3(caspase-3), and cleaved caspase-3, and EMT-associated proteins E-cadherin, MMP9, MMP2 and vimentin in HT-29 cells and mouse tumor tissues after the treatment of AC was determined by Western blot. The results showed that cell survival rate and the number of cells at proliferation stage decreased compared with those in the blank control group. The number of migrating and invading cells reduced and the number of apoptotic cells increased in the administration groups compared with those in the blank control group. As for the in vivo experiment, compared with the blank control group, the administration groups had small tumors with low mass and shrinkage of cells and karyopycnosis in the tumor tissue, indicating that the AC combination may improve EMT. In addition, the expression of Bcl2 and E-cadherin increased and the expression of Bax, caspase-3, cleaved caspase-3, MMP9, MMP2, and vimentin decreased in HT-29 cells and tumor tissues in each administration group. In summary, the AC combination can significantly inhibit the proliferation, invasion, migration, and EMT of HT-29 cells in vivo and in vitro and promote the apoptosis of colon cancer cells.
Humans ; Animals ; Mice ; Caspase 3 ; Matrix Metalloproteinase 2 ; Matrix Metalloproteinase 9 ; Vimentin ; HT29 Cells ; bcl-2-Associated X Protein ; Colonic Neoplasms ; Cell Proliferation

This study analyzes the impact of echinacoside(ECH) in the proliferation, metastasis and adriamycin(ADR) resistance of breast cancer(BC) MCF-7 cells via the modulation of aldo-keto reductase family 1 member 10(AKR1B10)/extracellular signal-regulated kinase(ERK) pathway. The chemical structure of ECH was firstly confirmed. MCF-7 cells were treated with different concentration(0, 10, 20, 40 μg·mL~(-1)) of ECH for 48 h. Western blot was used to analyze expression of AKR1B10/ERK pathway-associated proteins and cell counting kit-8(CCK-8) assay to determine cell viability. MCF-7 cells were collected and classified into control group, ECH group, ECH + Ov-NC group, and ECH + Ov-AKR1B10 group. Then Western blot was employed to analyze the expression of AKR1B10/ERK pathway-associated proteins. CCK-8 and 5-ethynyl-2'-deoxyuridine(EdU) assay were used to examine cell proliferation. Cell migration was appraised with scratch assay, Transwell assay, and Western blot. Eventually, MCF-7 cells were treated with ADR for 48 h to induce ADR resistance. Cell viability was tested by CCK-8 assay and cell apoptosis was estimated based on terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) assay and Western blot. Based on Protein Data Bank(PDB) and molecular docking, the binding affinity of ECH to AKR1B10 was assessed. Various doses of ECH decreased the expression of AKR1B10/ERK pathway-associated proteins in a dose-dependent manner and declined cell viability compared with the control group. Compared with the control group, 40 μg·mL~(-1) ECH blocked the AKR1B10/ERK pathway in MCF-7 cells and inhibited the proliferation, metastasis and ADR resistance of the cells. Compared with the ECH + Ov-NC group, ECH + Ov-AKR1B10 group showed the recovery of some biological behaviors of MCF-7 cells. ECH also targeted AKR1B10. ECH can inhibit the proliferation, metastasis, and ADR resistance of BC cells by blocking AKR1B10/ERK pathway.
Humans ; MCF-7 Cells ; Molecular Docking Simulation ; Sincalide ; Signal Transduction ; Neoplasms ; Aldo-Keto Reductases

This study explores the effect of apigenin(APG), oxymatrine(OMT), and APG+OMT on the proliferation of non-small cell lung cancer cell lines and the underlying mechanisms. Cell counting kit-8(CCK-8) assay was used to detect the vitality of A549 and NCI-H1975 cells, and colony formation assay to evaluate the colony formation ability of the cells. EdU assay was employed to examine the proliferation of NCI-H1975 cells. RT-qPCR and Western blot were performed to detect the mRNA and protein expression of PLOD2. Molecular docking was carried out to explore the direct action ability and action sites between APG/OMT and PLOD2/EGFR. Western blot was used to study the expression of related proteins in EGFR pathway. The viability of A549 and NCI-H1975 cells was inhibited by APG and APG+OMT at 20, 40, and 80 μmol·L~(-1) in a dose-dependent manner. The colony formation ability of NCI-H1975 cells was significantly suppressed by APG and APG+OMT. The mRNA and protein expression of PLOD2 was significantly inhibited by APG and APG+OMT. In addition, APG and OMT had strong binding activity with PLOD2 and EGFR. In APG and APG+OMT groups, the expression of EGFR and proteins in its downstream signaling pathways was significantly down-regulated. It is concluded that APG in combination with OMT could inhibit non-small lung cancer, and the mechanism may be related to EGFR and its downstream signaling pathways. This study lays a new theoretical basis for the clinical treatment of non-small cell lung cancer with APG in combination with OMT and provides a reference for further research on the anti-tumor mechanism of APG in combination with OMT.
Humans ; Carcinoma, Non-Small-Cell Lung ; Lung Neoplasms ; Apigenin ; Molecular Docking Simulation ; Alkaloids ; Quinolizines ; RNA, Messenger ; ErbB Receptors

This study aimed to explore the effect of Ganmai Dazao Decoction on the ethology of rats with posttraumatic stress disorder(PTSD) and study the related mechanism through the changes in magnetic resonance imaging and protein expression. Sixty rats were randomly divided into 6 groups, namely the normal group, the model group, the low(1 g·kg~(-1)), medium(2 g·kg~(-1)), and high-dose Ganmai Dazao Decoction groups(4 g·kg~(-1)), and the positive control group(intragastric administration with 10.8 mg·kg~(-1) of fluoxetine), with 10 rats in each group. Two weeks after inducing PTSD by single-prolonged stress(SPS) in rats, the positive control group was given fluoxetine hydrochloride capsule by gavage, the low, medium, and high-dose groups were given Ganmai Dazao Decoction by gavage, and both the normal group and the model group were given the same volume of normal saline by gavage, each for 7 days. The open field experiment, elevated cross elevated maze, forced swimming experiment, and new object recognition test were carried out for the behavioral test. Three rats in each group were selected to detect the expression of neuropeptide receptor Y1(NPY1R) protein in the hippocampus by Western blot. Then, the other three rats in each group were selected to use the 9.4T magnetic resonance imaging experiment to observe the overall structural changes in the brain region and the anisotropy fraction of the hippocampus. The results of the open field experiment showed that the total distance and central distance of rats in the model group were significantly lower than those in the normal group, and the total distance and central distance of rats in the middle and high-dose Ganmai Dazao Decoction groups were higher than those in the model group. The results of the elevated cross maze test showed that medium and high-dose Ganmai Dazao Decoction remarkably increased the number of open arm entries and the residence time of open arm of rats with PTSD. The results of the forced swimming experiment showed that the immobility time in the water of the model group rats was significantly higher than that of the normal group, and Ganmai Dazao Decoction hugely reduced the immobility time in the water of rats with PTSD. The results of the new object recognition test showed that Ganmai Dazao Decoction significantly increased the exploration time of new objects and familiar objects in rats with PTSD. The results of Western blot showed that Ganmai Dazao Decoction significantly reduced the expression of NYP1R protein in the hippocampus of rats with PTSD. The 9.4T magnetic resonance examination found that there was no significant difference in the structural image among the groups. In the functional image, the fractional anisotropy(FA value) of the hippocampus in the model group was significantly lower than that in the normal group. The FA value of the hippocampus in the middle and high-dose Ganmai Dazao Decoction groups was higher than that in the model group. Ganmai Dazao Decoction reduces the injury of hippocampal neurons by inhibiting the expression of NYP1R in the hippocampus of rats with PTSD, thereby improving the nerve function injury of rats with PTSD and playing a neuroprotective role.
Animals ; Rats ; Ethology ; Stress Disorders, Post-Traumatic ; Fluoxetine ; Hippocampus ; Maze Learning

This paper aimed to study the effect of Erjing Pills on the improvement of neuroinflammation of rats with Alzheimer's di-sease(AD) induced by the combination of D-galactose and Aβ_(25-35) and its mechanism. SD rats were randomly divided into a sham group, a model control group, a positive drug group(donepezil, 1 mg·kg~(-1)), an Erjing Pills high-dose group(9.0 g·kg~(-1)), and an Erjing Pills low-dose group(4.5 g·kg~(-1)), with 14 rats each group. To establish the rat model of AD, Erjing Pills were intragastrically administrated to rats for 5 weeks after 2 weeks of D-galactose injection. D-galactose was intraperitoneally injected into rats for 3 weeks, and then Aβ_(25-35) was injected into the bilateral hippocampus. The new object recognition test was used to evaluate the learning and memory ability of rats after 4 weeks of intragastric administration. Tissues were acquired 24 h after the last administration. The immunofluorescence method was used to detect the activation of microglia in the brain tissue of rats. The positive expressions of Aβ_(1-42) and phosphory protein Tau~(404)(p-Tau~(404)) in the CA1 area of the hippocampus were detected by immunohistochemistry. The levels of inflammatory factors interleukin-1β(IL-1β), tumor necrosis factor-α(TNF-α), and interleukin-6(IL-6) in the brain tissue were determined by enzyme-linked immunosorbent assay(ELISA). Toll-like receptor 4(TLR4)/nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptors 3(NLRP3) pathway-associated proteins in the brain tissue were determined by Western blot. The results showed that as compared with the sham group, the new object recognition index of rats in the model control group decreased significantly, the deposition of Aβ_(1-42) and p-Tau~(404) positive protein in the hippocampus increased significantly, and the levels of microglia activation increased significantly in the dentate gyrus. The levels of IL-1β, TNF-α, and IL-6 in the hippocampus of the model control group increased significantly, and the expression levels of TLR4, p-NF-κB p65/NF-κB p65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus increased significantly. Compared with the model control group, the Erjing Pill groups enhanced the new object recognition index of rats, decreased the deposition of Aβ_(1-42) and the expression of p-Tau~(404) positive protein in the hippocampus, inhibited the activation of microglia in the dentate gyrus, reduced the levels of inflammatory factors IL-1β, TNF-α, and IL-6 in the hippocampus, and down-regulated the expression levels of TLR4, p-NF-κB P65/NF-κB P65, p-IκBα/IκBα, and NLRP3 proteins in the hippocampus. In conclusion, Erjing Pills can improve the learning and memory ability of the rat model of AD presumably by improving the activation of microglia, reducing the expression levels of neuroinflammatory factors IL-1β, TNF-α, and IL-6, inhibiting the TLR4/NF-κB/NLRP3 neuroinflammation pathway, and decreasing hippocampal deposition of Aβ and expression of p-Tau, thereby restoring the hippocampal morphological structure.
Animals ; Rats ; Rats, Sprague-Dawley ; NF-kappa B ; NF-KappaB Inhibitor alpha ; NLR Family, Pyrin Domain-Containing 3 Protein ; Galactose ; Interleukin-6 ; Neuroinflammatory Diseases ; Toll-Like Receptor 4 ; Tumor Necrosis Factor-alpha

This study aimed to explore the potential mechanism of Berberis atrocarpa Schneid. anthocyanin against Alzheimer's disease(AD) based on network pharmacology, molecular docking technology, and in vitro experiments. Databases were used to screen out the potential targets of the active components of B. atrocarpa and the targets related to AD. STRING database and Cytoscape 3.9.0 were adopted to construct a protein-protein interaction(PPI) network and carry out topological analysis of the common targets. Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analyses were performed on the target using the DAVID 6.8 database. Molecular docking was conducted to the active components and targets related to the nuclear factor kappa B(NF-κB)/Toll-like receptor 4(TLR4) pathway. Finally, lipopolysaccharide(LPS) was used to induce BV2 cells to establish the model of AD neuroinflammation for in vitro experimental validation. In this study, 426 potential targets of active components of B. atrocarpa and 329 drug-disease common targets were obtained, and 14 key targets were screened out by PPI network. A total of 623 items and 112 items were obtained by GO functional enrichment analysis and KEGG pathway enrichment analysis, respectively. Molecular docking results showed that NF-κB, NF-κB inhibitor(IκB), TLR4, and myeloid differentiation primary response 88(MyD88) had good binding abilities to the active components, and malvidin-3-O-glucoside had the strongest binding ability. Compared with the model group, the concentration of nitric oxide(NO) decreased at different doses of malvidin-3-O-glucoside without affecting the cell survival rate. Meanwhile, malvidin-3-O-glucoside down-regulated the protein expressions of NF-κB, IκB, TLR4, and MyD88. This study uses network pharmacology and experimental verification to preliminarily reveal that B. atrocarpa anthocyanin can inhibit LPS-induced neuroinflammation by regulating the NF-κB/TLR4 signaling pathway, thereby achieving the effect against AD, which provides a theoretical basis for the study of its pharmacodynamic material basis and mechanism.
NF-kappa B ; Alzheimer Disease ; Network Pharmacology ; Anthocyanins ; Berberis ; Lipopolysaccharides ; Molecular Docking Simulation ; Myeloid Differentiation Factor 88 ; Neuroinflammatory Diseases ; Toll-Like Receptor 4 ; I-kappa B Proteins

This study aimed to identify the direct pharmacological targets of Jingfang Granules in treating infectious pneumonia via "target fishing" strategy. Moreover, the molecular mechanism of Jingfang Granules in treating infectious pneumonia was also investigated based on target-related pharmacological signaling pathways. First, the Jingfang Granules extract-bound magnetic nanoparticles were prepared, which were incubated with lipopolysaccharide(LPS)-induced mouse pneumonia tissue lysates. The captured proteins were analyzed by high-resolution mass spectrometry(HRMS), and the target groups with specific binding to the Jingfang Granules extract were screened out. Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis was used to identify the target protein-associated signaling pathways. On this basis, the LPS-induced mouse model of infectious pneumonia was established. The possible biological functions of target proteins were verified by hematoxylin-eosin(HE) staining and immunohistochemical assay. A total of 186 Jingfang Granules-specific binding proteins were identified from lung tissues. KEGG pathway enrichment analysis showed that the target protein-associated signaling pathways mainly included Salmonella infection, vascular and pulmonary epithelial adherens junction, ribosomal viral replication, viral endocytosis, and fatty acid degradation. The target functions of Jingfang Granules were related to pulmonary inflammation and immunity, pulmonary energy metabolism, pulmonary microcirculation, and viral infection. Based on the in vivo inflammation model, Jingfang Granules significantly improved the alveolar structure of the LPS-induced mouse model of infectious pneumonia and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6). Meanwhile, Jingfang Gra-nules significantly up-regulated the expressions of key proteins of mitochondrial function COX Ⅳ and ATP, microcirculation-related proteins CD31 and Occludin, and proteins associated with viral infection DDX21 and DDX3. These results suggest that Jingfang Gra-nules can inhibit lung inflammation, improve lung energy metabolism and pulmonary microcirculation, resist virus infection, thus playing a protective role in the lung. This study systematically explains the molecular mechanism of Jingfang Granules in the treatment of respiratory inflammation from the perspective of target-signaling pathway-pharmacological efficacy, thereby providing key information for clinical rational use of Jingfang Granules and expanding potential pharmacological application.
Animals ; Mice ; Lipopolysaccharides ; Pneumonia ; Inflammation ; Anti-Infective Agents ; Biological Assay ; Disease Models, Animal ; Interleukin-6

This study used bioinformatics analysis to screen out key genes involved in the transformation of idiopathic membranous nephropathy to end-stage renal disease and to predict targeted Chinese herbs and medicines and active ingredients with preventive and curative effects. The GSE108113 microarray of idiopathic membranous nephropathy and GSE37171 microarray of were downloaded from the comprehensive gene expression database, and 8 homozygous differentially expressed genes for the transformation of idiopathic membranous nephropathy into end-stage renal disease of were screened out by R software. GraphPad Prism was used to verify the expression of homozygous differentially expressed genes in GSE115857 microarray of idiopathic membranous nephropathy and GSE66494 microarray of chronic kidney disease, and 7 key genes(FOS, OGT, CLK1, TIA1, TTC14, CHORDC1, and ANKRD36B) were finally obtained. The Gene Ontology(GO) analysis was performed. There were 209 functions of encoded proteins, mainly involved in regulation of RNA splicing, cytoplasmic stress granule, poly(A) binding, etc. Thirteen traditional Chinese medicines with the effect of preventing the transformation of idiopathic membranous nephropathy to end-stage renal disease were screened out from Coremine Medical database, including Ginseng Radix et Rhizoma, Lycopi Herba, and Gardeniae Fructus, which were included in the Chinese Pharmacopoeia(2020 edition). The active ingredient quercetin mined from Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) had ability to dock with the key gene FOS-encoded protein molecule, which provided targets and research ideas for the development of new traditional Chinese medicines.
Humans ; Medicine, Chinese Traditional ; Glomerulonephritis, Membranous ; Kidney Failure, Chronic ; Renal Insufficiency, Chronic ; Computational Biology

Children's fever is often accompanied by food accumulation. Traditional Chinese medicine believes that removing food stagnation while clearing heat of children can effectively avoid heat damage. To systematically evaluate the efficacy of Xiaoer Chiqiao Qingre Granules(XRCQ) in clearing heat and removing food accumulation and explore its potential mechanism, this study combined suckling SD rats fed with high-sugar and high-fat diet with injection of carrageenan to induce rat model of fever and food accumulation. This study provided references for the study on the pharmacodynamics and mechanism of XRCQ. The results showed that XRCQ effectively reduced the rectal temperature of suckling rats, improved the inflammatory environment such as the content of interleukin-1β(IL-1β), interleukin-2(IL-2), interferon-γ(IFN-γ), white blood cells, and monocytes. XRCQ also effectively repaired intestinal injury and enhanced intestinal propulsion function. According to the confirmation of its efficacy of clearing heat, the thermolytic mechanism of XRCQ was further explored by non-targeted and targeted metabolomics methods based on LTQ-Orbitrap MS/MS and UPLC-QQQ-MS/MS. Non-target metabolomics analysis of brain tissue samples was performed by QI software combined with SIMCA-P software, and 22 endogenous metabolites that could be significantly regulated were screened out. MetaboAnalyst pathway enrichment results showed that the intervention mechanism was mainly focused on tyrosine metabolism, tricarboxylic acid cycle, inositol phosphate metabolism, and other pathways. At the same time, the results of targeted metabolomics of brain tissue samples showed that XRCQ changed the vitality of digestive system, and inhibited abnormal energy metabolism and inflammatory response, playing a role in clearing heat and removing food stagnation from multiple levels.
Animals ; Rats ; Rats, Sprague-Dawley ; Hot Temperature ; Tandem Mass Spectrometry ; Metabolomics ; Food ; Fever ; Interferon-gamma