This study aims to investigate the pathogenesis of chronic heart failure based on ferroptosis-mediated oxidative stress and predict the targets of Shenfu Injection in treating chronic heart failure. A rat model of chronic heart failure was established by the isoproterenol induction method. According to the random number table method, the modeled rats were assigned into three groups: a model group, a Shenfu Injection group, and a ferrostatin-1(ferroptosis inhibitor) group. In addition, a normal group was designed. After 15 days of intervention, the cardiac mass index and left ventricular mass index were determined. Echocardiography was employed to eva-luate the cardiac function. Hematoxylin-eosin staining and Masson staining were employed to reveal the pathological changes and fibrosis of the heart, and Prussian blue staining to detect the aggregation of iron ions in the myocardial tissue. Transmission electron microscopy was employed to observe the mitochondrion ultrastructure in the myocardial tissue. Colorimetry was adopted to measure the levels of iron metabolism, lipid peroxidation, and antioxidant indicators. Flow cytometry was employed to measure the content of lipid-reactive oxygen species(ROS) and the fluorescence intensity of ROS. Western blot and RT-qPCR were employed to determine the protein and mRNA levels, respectively, of ferroptosis-related factors in the myocardial tissue. The results showed that the rats in the model group had reduced cardiac function, elevated levels of total iron and Fe~(2+), lowered level of glutathione(GSH), increased malondialdehyde(MDA), decreased superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px), and rising levels of ROS and lipid-ROS. In addition, the model group showed fibrous tissue hyperplasia with inflammatory cell infiltration and myocardial fibrosis, iron ion aggregation, and characteristic mitochondrial changes specific for iron death. Moreover, the model group showcased upregulated protein and mRNA levels of p53 and COX2 and downregulated protein and mRNA levels of GPX4, FTH1, SLC7A11, and Nrf2 in the myocardial tissue. The intervention with Shenfu Injection significantly improved the cardiac function, recovered the iron metabolism, lipid peroxidation, and antioxidant indicators, decreased iron deposition, improved mitochondrial structure and function, and alleviated inflammatory cell infiltration and fibrosis. Furthermore, Shenfu Injection downregulated the mRNA and protein levels of p53 and COX2 and upregulated the mRNA and protein levels of GPX4, FTH1, SLC7A11, and Nrf2 in the myocardial tissue. Shenfu Injection can improve the cardiac function by regulating iron metabolism, inhibiting ferroptosis, and reducing oxidative stress injury.
Animals ; Rats ; Antioxidants ; Reactive Oxygen Species ; Cyclooxygenase 2 ; Ferroptosis ; NF-E2-Related Factor 2 ; Tumor Suppressor Protein p53 ; Heart Failure/genetics* ; Oxidative Stress ; Chronic Disease ; Glutathione ; Fibrosis ; Iron ; RNA, Messenger ; Lipids

OBJECTIVE: To explore the potential molecular mechanism of tetrahydropalmatine (THP) on acute myocardial ischemia (AMI). METHODS: First, the target genes of THP and AMI were collected from SymMap Database, Traditional Chinese Medicine Database and Analysis Platform, and Swiss Target Prediction, respectively. Then, the overlapping target genes between THP and AMI were evaluated for Grene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction network analysis. The binding affinity between the protein and THP was assessed by molecular docking. Finally, the protective effects of THP on AMI model and oxygen and glucose deprivation (OGD) model of H9C2 cardiomyocyte were explored and the expression levels of target genes were detected by RT-qPCR in vivo and in vitro. RESULTS: MMP9, PPARG, PTGS2, SLC6A4, ESR1, JAK2, GSK3B, NOS2 and AR were recognized as hub genes. The KEGG enrichment analysis results revealed that the potential target genes of THP were involved in the regulation of PPAR and hormone pathways. THP improved the cardiac function, as well as alleviated myocardial cell damage. Furthermore, THP significantly decreased the RNA expression levels of MMP9, PTGS2, SLC6A4, GSK3B and ESR1 (P<0.05, P<0.01) after AMI. In vitro, THP significantly increased H9C2 cardiomyocyte viability (P<0.05, P<0.01) and inhibited the RNA expression levels of PPARG, ESR1 and AR (P<0.05, P<0.01) in OGD model. CONCLUSIONS THP could improve cardiac function and alleviate myocardial injury in AMI. The underlying mechanism may be inhibition of inflammation, the improvement of energy metabolism and the regulation of hormones.
Humans ; Matrix Metalloproteinase 9 ; Network Pharmacology ; Cyclooxygenase 2 ; Molecular Docking Simulation ; PPAR gamma ; Myocardial Ischemia/genetics* ; Glucose ; RNA ; Drugs, Chinese Herbal/therapeutic use* ; Serotonin Plasma Membrane Transport Proteins

This study aimed to investigate the intervention effect and mechanism of Xiaoyao Kangai Jieyu Recipe(XKJR) on hip-pocampal microglia and neuronal damage in mice with breast cancer related depression. The mouse model of breast cancer related depression was established by inoculation of 4T1 breast cancer cells in axilla and subcutaneous injection of corticosterone(30 mg·kg~(-1)). The successfully modeled mice were randomly divided into a model group, a positive drug group(capecitabine 60 mg·kg~(-1)+fluoxetine 19.5 mg·kg~(-1)), and XKJR group(19.5 mg·kg~(-1) crude drug), with 6 in each group. Another 6 normal mice were taken as a normal group. The administration groups were given corresponding drugs by gavage, while the normal and model groups were given an equal volume of distilled water, once a day for 21 consecutive days. The depressive behavior of mice was assessed by glucose consumption test, open field test and novelty-suppressed feeding test. Hematoxylin and eosin(HE) staining and tumor suppression rate were used to evaluate the changes of axillary tumors. The mRNA expressions and the relative protein expressions of interleukin-1β(IL-1β), interleukin-18(IL-18), cyclooxyganese-2(COX-2) and glutamyl-prolyl-tRNA synthetase(EPRs) in the hippocampus of mice were determined by quantitative real-time polymerase chain reaction(qRT-PCR) and immunohistochemistry, respectively. Immunofluorescence was performed to detect the mean fluorescence intensity of CD11b, a marker of hippocampal microglia activation. Nissler staining and transmission electron microscopy were employed to observe the morphological changes and the ultramorphological changes of hippocampal neurons, respectively. The experimental results indicated that compared with the normal group, the model group had reduced glucose consumption and lowered number of total activities in open field test(P<0.05, P<0.01), prolonged first feeding latency in no-velty-suppressed feeding test(P<0.01), and significant depression-like behavior; the contents of IL-1β, IL-18, COX-2, and EPRs in hippocampus were increased(P<0.05, P<0.01), with hippocampal microglia activation and obvious neuronal damage. Compared with the model group, the positive drug group and the XKJR group presented an improvement in depressive behaviors, a decrease in the contents of IL-1β, IL-18, COX-2 and EPRs in hippocampus, and an alleviation in the activation of hippocampal microglia and neuronal damage; the tumor suppression rates of positive drug and XKJR were 40.32% and 48.83%, respectively, suggesting a lower tumor growth rate than that of the model group. In summary, XKJR may improve hippocampal microglia activation and neuronal damage in mice with breast cancer related depression through activating COX signaling pathway.
Mice ; Animals ; Depression/genetics* ; Interleukin-18 ; Cyclooxygenase 2/genetics* ; Hippocampus ; Glucose ; Neoplasms

Objective To investigate the effect of long intergenic non-coding RNA COX2 (lincRNA-COX2) on apoptosis and polarization of Listeria monocytogenes (Lm)-infected RAW264.7 cells. Methods RAW264.7 cells were cultured and divided into control group (uninfected cells), Lm infection group, negative control of small interfering RNA (si-NC) group, si-NC and Lm infection group, small interfering RNA of lincRNA-COX2 (si-lincRNA-COX2) group, si-lincRNA-COX2 and Lm infection group. RAW264.7 cells were infected with MOI=10 Lm for 6 hours, and then the inhibition efficiency of siRNA transfection was detected by fluorescence microscope and quantitative real-time PCR (qRT-PCR). The expression levels of cleaved-caspase-3(c-caspase-3), caspase-3, B-cell lymphoma-2 (Bcl2), Bcl2 associated X protein (BAX), arginase 1 (Arg1), inducible nitric oxide synthase (iNOS) were detected by Western blot analysis. Results c-caspase-3/caspase-3, BAX/Bcl2 and iNOS were significantly up-regulated, while the level of Arg1 was down-regulated in Lm-infected RAW264.7 cells compared with control group. LincRNA-COX2 knockdown inhibited the increase of protein levels for BAX/Bcl2, c-caspase-3/caspase-3 and iNOS in Lm-infected RAW264.7 cells, while the level of Arg1 in Lm-infected RAW264.7 cells was up-regulated. Conclusion Knockdown of lincRNA-COX2 can inhibit cell apoptosis and suppress the macrophage polarization into M1 type in Lm-infected RAW264.7 cells.
Apoptosis/genetics* ; bcl-2-Associated X Protein/metabolism* ; Caspase 3/metabolism* ; Cyclooxygenase 2/metabolism* ; Listeria monocytogenes/pathogenicity* ; Macrophages/microbiology* ; RNA, Long Noncoding/metabolism* ; RNA, Small Interfering/genetics* ; Animals ; Mice

To investigated the mechanisms underlying the effects of modified Kaixin San(MKXS) on improving memory and synaptic damage of Alzheimer's disease(AD) mouse model with conditional presenilin 1/2 conditional double knockout(PS cDKO). Specifically, 60 PS cDKO mice(3-3.5 months old) and their age-matched wild-type(WT) littermates were randomized into three groups: WT group(n=20), PS cDKO group(n=20), and PS cDKO+MKXS group(n=20). Mice in WT and PS cDKO groups were fed with standard chow and those in PS cDKO+MKXS group were given chow containing MKXS(at 2.55 g·kg~(-1)) for 60 days. Novel object reco-gnition task was employed to detect the recognition memory of mice, and Western blot to detect the protein levels of synapse-associated proteins in the hippocampus(HPC) of mice, such as NR1, NR2 A, NR2 B, p-αCaMKⅡ, tau, and p-tau. Microglial morphology in the HPC CA1 of mice was observed based on immunohistochemistry. Quantitative real time-PCR(qRT-PCR) was employed to detect the mRNA levels of the pro-inflammatory factors and synapse-associated proteins in the HPC of mice, including COX-2, iNOS, IL-1β, IL-6, TNF-α, PSD95, NR1, NR2 A, NR2 B, and MAP2. The protein levels of IL-1β, TNF-α, and IL-6 were tested by enzyme-linked immunosorbent assay(ELISA). The interaction between PSD95 and αCaMKⅡ and between PSD95 and p-αCaMKⅡ was tested by co-immunoprecipitation(Co-IP). The results showed that PS cDKO+MKXS demonstrated significantly higher preference index and recognition index of the new objects, lower protein level of p-tau(ser 396/404) and mRNA levels of COX-2, iNOS, TNF-α, IL-1β, and IL-6 in HPC, higher protein levels of NR1, NR2 A, NR2 B, and p-αCaMKⅡ and mRNA levels of NR1, NR2 A, NR2 B, PSD95, and MAP2, and stronger interaction of αCaMKⅡ with PSD95 and interaction of p-αCaMKⅡ with PSD95 than the PS cDKO group. Immunohistoche-mical staining showed that MKXS inhibited the activation of microglia. In conclusion, MKXS improves memory and synaptic damage in mice with AD by modulating αCaMKⅡ-PSD95 protein binding through inhibition of neuroinflammation.
Animals ; Mice ; Alzheimer Disease/genetics* ; Disks Large Homolog 4 Protein/metabolism* ; Neuroinflammatory Diseases ; Tumor Necrosis Factor-alpha/metabolism* ; Cyclooxygenase 2/metabolism* ; Interleukin-6/metabolism* ; Protein Binding ; Mice, Knockout ; Hippocampus/metabolism* ; Disease Models, Animal ; RNA, Messenger/metabolism*

The present study aimed to explore the regulatory targets and anti-inflammatory mechanism of Jingfang Mixture based on network pharmacology and animal tests. The active ingredients of Jingfang Mixture and the corresponding targets were screened out by the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP). Inflammation-related targets were searched from GeneCards and DisGeNET, and the targets of active ingredients of Jingfang Mixture against inflammation were obtained. The protein-protein interaction(PPI) network was analyzed by STRING and plotted. Gene ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were carried out based on DAVID. The results of network pharmacology showed 159 active ingredients and 276 targets of Jingfang Mixture and 664 inflammation-related targets were screened out, and 90 targets of active ingredients of Jingfang Mixture against inflammation were obtained. As revealed by the PPI network, protein kinase B1(AKT1), caspase-3(CASP3), interleukin-1β(IL1 B), prostaglandin-endoperoxide synthase 2(PTGS2), and tumor necrosis factor(TNF) might be the key proteins for the anti-inflammatory effect of Jingfang Mixture. KEGG enrichment analysis demonstrated the pathways involved TNF, nuclear factor-kappa B(NF-κB), and mitogen-activated protein kinase(MAPK). The anti-inflammatory effect of Jingfang Mixture was explored through the mouse model of urticaria. The results indicated that Jingfang Mixture could down-regulate the phosphorylation levels of p38 MAPK, extracellular regulated protein kinases(ERK1/2), and NF-κB. The present study revealed the anti-inflammatory effect of Jingfang Mixture with multi-component and multi-target characteristics, which is expected to provide a scientific basis and important support for further research, development, and application.
Animals ; Mice ; Anti-Inflammatory Agents/therapeutic use* ; Cyclooxygenase 2 ; Drugs, Chinese Herbal/therapeutic use* ; Inflammation/drug therapy* ; Medicine, Chinese Traditional ; Molecular Docking Simulation ; Network Pharmacology ; NF-kappa B/genetics*

In the present study, we investigated anti-inflammatory effect of Cardamine komarovii flower (CKF) on lipopolysaccharide (LPS)-induced acute lung injury (ALI). We determined the effect of CKF methanolic extracts on LPS-induced pro-inflammatory mediators NO and prostaglandin E2 (PGE2), production of pro-inflammatory cytokines (IL-1β, TNF-α, and IL-6), and related protein expression levels of MyD88/TRIF signaling pathways in peritoneal macrophages (PMs). Nuclear translocation of NF-κB-p65 was analyzed by immunofluorescence. For the in vivo experiments, an ALI model was established to detect the number of inflammatory cells and inflammatory factors (IL-1β, TNF-α, and IL-6) in bronchoalveolar lavage fluid (BALF) of mice. The pathological damage in lung tissues was evaluated through H&E staining. Our results showed that CKF can decrease the production of inflammatory mediators, such as NO and PGE2, by inhibiting their synthesis-related enzymes iNOS and COX-2 in LPS-induced PMs. In addition, CKF can downregulate the mRNA levels of IL-1β, TNF-α, and IL-6 to inhibit the production of inflammatory factors. Mechanism studies indicated that CKF possesses a fine anti-inflammatory effect by regulating MyD88/TRIF dependent signaling pathways. Immunocytochemistry staining showed that the CKF extract attenuates the LPS-induced translocation of NF-kB p65 subunit in the nucleus from the cytoplasm. In vivo experiments revealed that the number of inflammatory cells and IL-1β in BALF of mice decrease after CKF treatment. Histopathological observation of lung tissues showed that CKF can remarkably improve alveolar clearance and infiltration of interstitial and alveolar cells after LPS stimulation. In conclusion, our results suggest that CKF inhibits LPS-induced inflammatory response by inhibiting the MyD88/TRIF signaling pathways, thereby protecting mice from LPS-induced ALI.
Acute Lung Injury ; chemically induced ; drug therapy ; genetics ; metabolism ; Adaptor Proteins, Vesicular Transport ; genetics ; metabolism ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; chemistry ; Cardamine ; chemistry ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Flowers ; chemistry ; Humans ; Lipopolysaccharides ; adverse effects ; Male ; Mice ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; NF-kappa B ; genetics ; metabolism ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Plant Extracts ; administration & dosage ; chemistry ; Signal Transduction ; drug effects ; Tumor Necrosis Factor-alpha ; genetics ; metabolism

Dendrobii Caulis (DC), named 'Shihu' in Chinese, is a precious herb in traditional Chinese medicine. It is widely used to nourish stomach, enhance body fluid production, tonify "Yin" and reduce heat. More than thirty Dendrobium species are used as folk medicine. Some compounds from DC exhibit inhibitory effects on macrophage inflammation. In the present study, we compared the anti-inflammatory effects among eight Dendrobium species. The results provided evidences to support Dendrobium as folk medicine, which exerted its medicinal function partially by its inhibitory effects on inflammation. To investigate the anti-inflammatory effect of Dendrobium species, mouse macrophage cell line RAW264.7 was activated by lipopolysaccharide. The nitric oxide (NO) level was measured using Griess reagent while the pro-inflammatory cytokines were tested by ELISA. The protein expressions of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2) and mitogen-activated protein kinases (MAPKs) phosphorylation were evaluated by Western blotting analysis. Among the eight Dendrobium species, both water extracts of D. thyrsiflorum B.S.Williams (DTW) and D. chrysotoxum Lindl (DCHW) showed most significant inhibitory effects on NO production in a concentration-dependent manner. DTW also significantly reduced TNF-α, MCP-1, and IL-6 production. Further investigations showed that DTW suppressed iNOS and COX-2 expression as well as ERK and JNK phosphorylation, suggesting that the inhibitory effects of DTW on LPS-induced macrophage inflammation was through the suppression of MAPK pathways. In conclusion, D. thyrsiflorum B.S.Williams was demonstrated to have potential to be used as alternative or adjuvant therapy for inflammation.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Cyclooxygenase 2 ; genetics ; Cytokines ; metabolism ; Dendrobium ; chemistry ; Gene Expression Regulation, Enzymologic ; drug effects ; Inflammation ; chemically induced ; drug therapy ; Lipopolysaccharides ; Macrophages ; drug effects ; enzymology ; Mice ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Nitric Oxide ; analysis ; Nitric Oxide Synthase Type II ; genetics ; Phosphorylation ; drug effects ; Plant Extracts ; pharmacology ; RAW 264.7 Cells ; Signal Transduction ; drug effects

The present study was designed to synthesize 2-Cyano-3, 12-dioxooleana-1, 9(11)-en-28-oate-13β, 28-olide (1), a lactone derivative of oleanolic acid (OA) and evaluate its anti-inflammatory activity. Compound 1 significantly diminished nitric oxide (NO) production and down-regulated the mRNA expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Further in vivo studies in murine model of LPS-induced acute lung injury (ALI) showed that 1 possessed more potent protective effects than the well-known anti-inflammatory drug dexamethasone by inhibiting myeloperoxidase (MPO) activity, reducing total cells and neutrophils, and suppressing inflammatory cytokines expression, and thus ameliorating the histopathological conditions of the injured lung tissue. In conclusion, compound 1 could be developed as a promising anti-inflammatory agent for intervention of LPS-induced ALI.
Acute Lung Injury ; drug therapy ; genetics ; immunology ; Animals ; Anti-Inflammatory Agents ; administration & dosage ; chemical synthesis ; Bronchoalveolar Lavage Fluid ; immunology ; Cyclooxygenase 2 ; genetics ; immunology ; Female ; Humans ; Interleukin-1beta ; genetics ; immunology ; Interleukin-6 ; genetics ; immunology ; Lipopolysaccharides ; adverse effects ; Lung ; drug effects ; immunology ; Macrophages ; drug effects ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Neutrophils ; drug effects ; immunology ; Oleanolic Acid ; administration & dosage ; analogs & derivatives ; chemical synthesis ; Peroxidase ; genetics ; immunology ; RAW 264.7 Cells ; Tumor Necrosis Factor-alpha ; genetics ; immunology

The etiology and pathogenesis of moyamoya disease (MMD) remain elusive. Some inflammatory proteins, such as cyclooxygenase (COX)-2, are believed to be implicated in the development of MMD. So far, the relationship between COX-2 and MMD is poorly understood and reports on the intracranial vessels of MMD patients are scanty. In this study, tiny pieces of middle cerebral artery (MCA) and superficial temporal artery (STA) from 13 MMD patients were surgically harvested. The MCA and STA samples from 5 control patients were also collected by using the same technique. The expression of COX-2 was immunohistochemically detected and the average absorbance (A) of positively-stained areas was measured. High-level COX-2 expression was found in all layers of the MCA samples from all 5 hemorrhagic MMD patients, while positive but weak expression of COX-2 was observed only in the endothelial layer of the MCA samples from most ischemic MMD patients (6/8, 75%). The average A values of COX-2 in the hemorrhagic MMD patients were substantially higher than those in their ischemic counterparts (t=4.632, P=0.001). There was no significant difference in the COX-2 expression among the "gender" groups, or "radiographic grade" groups, or "lesion location" groups (P>0.05 for all). The COX-2 expression was detected neither in the MCA samples from the controls nor in all STA specimens. Our results suggested that COX-2 was up-regulated in the MCA of MMD patients, especially in hemorrhagic MMD patients. We are led to speculate that COX-2 may be involved in the pathogenesis of MMD and even contribute to the hemorrhagic stroke of MMD patients.
Adult ; Case-Control Studies ; Cyclooxygenase 2 ; genetics ; metabolism ; Female ; Humans ; Intracranial Hemorrhages ; enzymology ; etiology ; Male ; Middle Aged ; Middle Cerebral Artery ; metabolism ; Moyamoya Disease ; complications ; enzymology