This study aimed to explore the protective effect of Reduning Injection(RDN) on mice infected by influenza virus A/PR/8(PR8) and its immune regulatory roles during viral infection. In in vivo experiments, female C57 BL/6 mice were randomly divided into phosphate buffered saline(PBS) group, PR8-infected group, oseltamivir treatment group(OSV) and RDN treatment group. After 2 h of PR8 infection, mice in the oseltamivir group were gavaged with oseltamivir 30 mg·kg~(-1), and those in the RDN treatment group were injected intraperitoneally with RDN 1.5 mL·kg~(-1)once per day for seven consecutive days. The body weight of mice in each group was recorded at the same time every morning for 16 consecutive days. The line chart of body weight change was created to analyze the protective effect of RDN on flu-infected mice. The relative mRNA expression of different cytokines(IL-6, TNF-α, MCP-1, IL-1β, MIP-2, IP-10 and IL-10) in lung samples of flu-infected mice was detected by PCR. Flow cytometry was utilized to analyze the composition of immune cells of mouse BALF samples on day 5 after infection. Mouse macrophage cell line RAW264.7 was planted and treated by different concentrations of RDN(150, 300, 600 μg·mL~(-1)) for 24 h or 48 h, and cell proliferation was detected by CCK-8 assay. RAW264.7 cells and mouse primary peritoneal macrophages were stimulated with synthetic single stranded RNA(R837), which elicited the inflammatory response by mimicking the infection of single-stranded RNA viruses. The expression of cytokines and chemokines in the supernatants of above culture system was detected by ELISA and qPCR. On days 4, 5, 6, 7 and 15 after infection, the body weight loss of mice in the RDN treatment group was alleviated compared with that of PR8-infected mice(P<0.05). RDN treatment obviously reduced lung index and the production of IL-6, TNF-α, MCP-1 and MIP-2 in lung tissues of flu-infected mice(P<0.05). The proportions of macrophages, neutrophils and T cells in mouse BALF samples were analyzed by flow cytometry, and compared with PR8-infected mice, RDN decreased the proportion of macrophages in BALF of flu-infected mice(P<0.05), and the proportion of T cells was recovered dramatically(P<0.001). In CCK-8 assay, the concentrations of RDN(150, 300, 600 μg·mL~(-1)) failed to cause cytotoxicity to RAW264.7 cells. In addition, RDN lowered the expression of inflammatory cytokines such as IL-6, TNF-α,MCP-1, IL-1β, RANTES, and IP-10 and even anti-inflammatory cytokine IL-10 in R837-induced macrophages. RDN reduced the infiltration of inflammatory macrophages and the production of excessive inflammatory cytokines, alleviated the body weight loss of flu-infected mice. What's more, RDN restored the depletion of T cells, which might prevent secondary infection and deteriorative progression of the disease. Taken together, RDN may inhibit cytokine production and therefore down-regulate cytokine storm during the infection of influenza virus.
Animals ; Anti-Inflammatory Agents/pharmacology* ; Body Weight ; Chemokine CCL5/pharmacology* ; Chemokine CXCL10/pharmacology* ; Cytokine Release Syndrome ; Cytokines/genetics* ; Drugs, Chinese Herbal ; Female ; Imiquimod/pharmacology* ; Interleukin-10 ; Interleukin-6 ; Lung ; Mice ; Mice, Inbred C57BL ; Oseltamivir/pharmacology* ; Phosphates/pharmacology* ; RNA ; RNA, Messenger ; Sincalide/pharmacology* ; Tumor Necrosis Factor-alpha/genetics* ; Weight Loss

OBJECTIVEA study was conducted to investigate the effects of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS) on the expression of regulated upon activation normal T-cell expressed and secreted (RANTES) and fractalkine in human umbilical vein endothelial cells (HUVECs).

METHODSHUVECs were incubated with different concentrations of Pg-LPS (200, 500, and 1000 ng x mL(-1)) for 1, 6, 12, and 24 h, respectively. Then real time quantitative polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent method (ELISA) were adopted to detect the protein levels and mRNA levels of RANTES and fractalkine.

RESULTSThe RANTES protein levels and mRNA levels, as well as fractalkine mRNA levels, were significantly higher in all experimental groups of 1, 6, and 12 h than in the control group (P<0.05), except the expression of RANTES mRNA in 200 ng x mL(-1) group of 12 h and RANTES protein in 200 ng x mL(-1) group of 1 h. The expression levels of RANTES mRNA and fractalkine mRNA were highest in 1000 ng x mL(-1) group of 6 h and were 4.88- and 6.20-fold higher, respectively, than those in the control group. The expression levels of RANTES protein, mRNA, and fractalkine mRNA decreased 6 h after stimulation, and were significantly higher than those in the control group (P<0.05) in the RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis 500 ng x mL(-1) group of 24 h. There was a significant difference between the expression of fractalkine mRNA in 1000 ng x mL(-1) group of 6 and 12 h than in the control group (P<0.05).

CONCLUSIONPg-LPS infection might up-regulate the expression of RANTES and fractalkine in HUVEC, and such expression is important in the development of atherosclerosis.

Atherosclerosis ; Cells, Cultured ; Chemokine CCL5 ; genetics ; metabolism ; Chemokine CX3CL1 ; analysis ; genetics ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; pharmacology ; Porphyromonas gingivalis ; immunology ; isolation & purification ; RNA, Messenger ; analysis ; Reverse Transcriptase Polymerase Chain Reaction ; Up-Regulation

Marsdenia tenacissima, a traditional Chinese medicine, is long been used to treat various diseases including asthma, cancer, trachitis, tonsillitis, pharyngitis, cystitis, and pneumonia. Although Marsdenia tenacissima has been demonstrated to have strong anti-tumor effects against primary tumors, its effect on cancer metastasis remains to be defined, and the molecular mechanism underlying the anti-metastatic effect is unknown. In the present study, we investigated the effects of XAP (an extract of Marsdenia tenacissima) on A549 lung cancer cell migration and explored the role of CCR5-CCL5 axis in the anti-metastatic effects of XAP. Our resutls showed that XAP inhibited A549 lung cancer cell migration and invasion in a dose-dependent manner. The protein levels of CCR5, but not CCR9 and CXCR4, were decreased by XAP. The secretion of CCL5, the ligand of CCR5, was reduced by XAP. XAP down-regulated Rho C expression and FAK phosphorylation. In conclusion, XAP inhibited A549 cell migration and invasion through down-regulation of CCR5-CCL5 axis, Rho C, and FAK.
A549 Cells ; Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Line, Tumor ; Cell Movement ; drug effects ; Chemokine CCL5 ; metabolism ; Focal Adhesion Kinase 1 ; metabolism ; Humans ; Lung Neoplasms ; Marsdenia ; chemistry ; Phosphorylation ; Plant Extracts ; pharmacology ; Receptors, CCR5 ; metabolism ; rho GTP-Binding Proteins ; metabolism ; rhoC GTP-Binding Protein

OBJECTIVETo investigate the preventive effects of Qiangzhi Decoction (, QZD) on influenza A pneumonia through inhibition of inflammatory cytokine storm in vivo and in vitro.

METHODSOne hundred ICR mice were randomly divided into the virus control, the Tamiflu control and the QZD high-, medium-, and low-dose groups. Mice were infected intranasally with influenza virus (H1N1) at 10 median lethal dose (LD50). QZD and Tamiflu were administered intragastrically twice daily from day 0 to day 7 after infection. The virus control group was treated with distilled water alone under the same condition. The number of surviving mice was recorded daily for 14 days after viral infection. The histological damage and viral replication and the expression of inflammatory cytokines were monitored. Additionally, the suppression capacity on the secretion of regulated on activation normal T cells expressed and secreted (RANTES) and tumor necrosis factor-α (TNF-α) in epithelial and macrophage cell-lines were evaluated.

RESULTSCompared with the virus control group, the survival rate of the QZD groups significantly improved in a dose-dependent manner (P<0.05), the viral titers in lung tissue was inhibited (P<0.05), and the production of inflammatory cytokines interferon-γ (IFN-γ), interleukin-6 (IL-6), TNF-α, and intercellular adhesion molecule-1 (ICAM-1) were suppressed (P<0.05). Meanwhile, the secretion of RANTETS and TNF-α by epithelial and macrophage cell-lines was inhibited with the treatment of QZD respectively in vitro (p<0.05) CONCLUSIONS: The preventive effects of QZD on influenza virus infection might be due to its unique cytokine inhibition mechanism. QZD may have significant therapeutic potential in combination with antiviral drugs.

Animals ; Cell Line ; Cell Survival ; drug effects ; Chemokine CCL5 ; metabolism ; Chemokines ; metabolism ; Cytokines ; metabolism ; Dogs ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Enzyme-Linked Immunosorbent Assay ; Hemagglutination, Viral ; drug effects ; Humans ; Inflammation ; pathology ; Influenza A Virus, H1N1 Subtype ; drug effects ; physiology ; Influenza A Virus, H1N2 Subtype ; drug effects ; Lung ; drug effects ; pathology ; Madin Darby Canine Kidney Cells ; Mice, Inbred ICR ; Orthomyxoviridae Infections ; complications ; pathology ; prevention & control ; Pneumonia ; complications ; pathology ; prevention & control ; Protective Agents ; pharmacology ; therapeutic use ; Survival Rate ; Tumor Necrosis Factor-alpha ; pharmacology

We evaluated the effect of cobalt chloride (CoCl2) on TNF-alpha and IFN-gamma-induced-inflammation and reactive oxygen species (ROS) in renal tubular epithelial cells (HK-2 cells). We treated HK-2 cells with CoCl2 before the administration of TNF-alpha/IFN-gamma. To regulate hemeoxygenase-1 (HO-1) expression, the cells were treated CoCl2 or HO-1 siRNA. CoCl2 reduced the generation of ROS induced by TNF-alpha/IFN-gamma. TNF-alpha/IFN-gamma-treated-cells showed an increase in the nuclear translocation of phosphorylated NF-kappaBp65 protein, the DNA-binding activity of NF-kappaBp50 and NF-kappaB transcriptional activity and a decrease in IkappaBalpha protein expression. These changes were restored by CoCl2. We noted an intense increase in monocyte chemoattractant protein-1 (MCP-1) and regulated on activation normal T cell expressed and secreted (RANTES) production in TNF-alpha/IFN-gamma-treated cells. We demonstrated that this effect was mediated through NF-kappaB signaling because an NF-kappaB inhibitor significantly reduced MCP-1 and RANTES production. CoCl2 effectively reduced MCP-1 and RANTES production. The expression of HO-1 was increased by CoCl2 and decreased by HO-1 siRNA. However, knockdown of HO-1 by RNA interference did not affect MCP-1 or RANTES production. We suggest that CoCl2 has a protective effect on TNF-alpha/IFN-gamma-induced inflammation through the inhibition of NF-kappaB and ROS in HK-2 cells. However, CoCl2 appears to act in an HO-1-independent manner.
Cell Line ; Chemokine CCL2/metabolism ; Chemokine CCL5/metabolism ; Cobalt/*pharmacology ; Epithelial Cells/cytology/metabolism ; Heme Oxygenase-1/antagonists & inhibitors/genetics/metabolism ; Humans ; *Inflammation ; Interferon-gamma/pharmacology ; Kidney Tubules, Proximal/cytology ; NF-kappa B/antagonists & inhibitors/genetics/*metabolism ; NF-kappa B p50 Subunit/genetics/metabolism ; Oxidative Stress/*drug effects ; Phosphorylation ; Protein Binding ; RNA Interference ; RNA, Small Interfering/metabolism ; Transcription Factor RelA/metabolism ; Tumor Necrosis Factor-alpha/pharmacology

OBJECTIVETo investigate the effect of curcumin on the injury in hippocampal neurons and the expression of regulated upon activation nonnal T-cell expressed and secreted (RANTES) in hippocamp during cerebral ischemia/reperfusion (I/R) in rats with spontaneous hypertension (SH).

METHODSMale Wistar-Kyoto (WKY) rats and spontaneous hypertension rats (SHR) were randomly divided into five groups (n = 6): sham group (W-Sham and S-Sham group), ischemia/reperfusion group (W-/R and S/R group), curcumin group (S-Cur group) . Each group was splitted into 5 subgroups of 3 h,12 h, 1 d, 3 d and 7 d according to the time interval before reperfusion. Global brain ischemia/reperfusion model was established by 4-VO method. Hematoxylin-eosin staining (HE staining) was used to observe the vertebral cell morphology in hippocampal CA1 region. Nissl staining was applied to detect the average density of cone cells in hippocampal CA1 region. The expression of RANTES in hippocamp was determined by ELISA. The behavior of the rats was evaluated at 7 days after reperfusion. Results: Compared with the sham group rats, the ability of learning and memory was significantly decreased in ischemia/reperfusion group rats, the number of injured neurons were greatly elevated , the protein expression levels of RANTES was significantly increased (P < 0.05). Compared with W-I/R group rats, the ability of learning and memory in S-I/R group rats was greatly reduced, the number of injured neurons increased extremely, the protein expression level of RANTES was significantly enhanced( P <0.05). The number of injured neurons declined significantly in S-Cur group rats, the ability to learn and remember of these rats was improved and the RANTES protein content decreased significantly (P < 0.05).

CONCLUSIONSHR are more susceptible to ischemia/reperfusion induced hippocampal neuronal injury which may be improved by curcu min. Its underlying mechanism is possibly associated with the inhibition of RANTES protein expression level.

Animals ; Brain Ischemia ; metabolism ; pathology ; physiopathology ; Chemokine CCL5 ; metabolism ; Cognition ; drug effects ; Curcumin ; pharmacology ; Hippocampus ; cytology ; metabolism ; pathology ; Hypertension ; metabolism ; pathology ; physiopathology ; Male ; Neurons ; drug effects ; metabolism ; pathology ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY ; Reperfusion Injury ; metabolism

The present study was undertaken to determine the effect of epigallocatechin gallate (EGCG) on lipopolysaccharide (LPS)-induced production of inflammatory chemokine regulated upon activation normal T cell expressed and secreted (RANTES) in human retinal endothelial cells (HRECs) and to explore the underlying regulatory mechanism. HRECs were stimulated with LPS in the presence or absence of EGCG at various concentrations (100, 50, 25, 12.5, 6.25 μmol/L). The optimum concentration of drug was determined by a real-time cell-electronic sensing (RT-CES) system, and MTS chromatometry was used to detect the toxicity of LPS and EGCG on HRECs. RANTES production in the culture supernatant was measured by ELISA. The expression levels of Akt and phosphorylated Akt were examined by Western blot assay. The result showed that LPS markedly stimulated RANTES secretion from HRECs. EGCG treatment significantly suppressed LPS-induced RANTES secretion in a dose-dependent manner. Furthermore, EGCG exhibited a dose-dependent inhibitory effect on LPS-induced phosphorylation of Akt. Taken together, our data suggest that EGCG suppresses LPS-induced RANTES secretion, possibly via inhibiting Akt phosphorylation in HRECs.
Catechin ; analogs & derivatives ; pharmacology ; Cells, Cultured ; Chemokine CCL5 ; metabolism ; Endothelial Cells ; metabolism ; Humans ; Lipopolysaccharides ; Phosphorylation ; Proto-Oncogene Proteins c-akt ; metabolism ; Retina ; cytology

OBJECTIVETo investigate the effect of triptolide (TPL) on the renal tissue of diabetic rats and its possible mechanisms.

METHODSSD rats were randomly divided into the normal control group (as the normal group), the diabetic model group (the model group), the low dose TPL treatment group (the low dose TPL group, TPL 0.2 mg/kg by gastrogavage), the high dose TPL treatment group (the high dose TPL group, TPL 0.4 mg/kg by gastrogavage). Equal volume of normal saline was given to rats in the normal group and the model group. Five rats were randomly selected from each group at week 4, 8, and 12 of the experiment to detect body weight, kidney weight, 24 h urinary albumin (24 h UAL), plasma glucose (FBG), total cholesterol (TC), total triglyeride (TG), alanine aminotransferase (ALT), aspartate aminotransferase (AST), white blood cell (WBC), and hemoglobin A1c (HbA1c). The mRNA and protein expression of regulated upon activation normal T-cell expressed and secreted (RANTES) in the renal tissue was assessed by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme linked immunosorbent assay (ELISA). The renal tissue was pathologically stained by HE, PAS, and Masson staining. The glomerular and renal tubular interstitial lesions were observed at each time point. The glomerular sclerosis index (GSI) was observed by PAS staining, and the renal interstitial filrosis index (RIFI) was calcutated.

RESULTSCompared with the same group at week 4, the expression of 24 h UAL, RANTES, GSI, and RIFI at week 12 significantly decreased in two TPL groups (P <0.01). Compared with the same group at week 8, the expression of 24 h UAL, RANTES, GSI, and RIFI at week 12 also significantly decreased in the two TPL groups (P <0. 05, P <0.01). Compared with the normal group, body weight and the kidney weight obviously decreased at week 4, 8, and 12 in the model group (P <0. 01); 24 h UAL, FBG, TG, TC, HbA1c, RANTES, GSI, and RIFI were obviously elevated (P <0.01). Compared with the model group, 24 h UAL, RANTES, GSI, and RIFI also decreased in the two TPL treatment groups (P <0.01). Compared with the low dose TPL group, they were attenuated in the high dose TPL group (P <0. 05, P <0. 01).

CONCLUSIONTPL could not only inhibit the over-expression of RANTES, but also improve the glomerular sclerosis and renal interstitial fibrosis in the renal tissue of diabetic rats.

Animals ; Chemokine CCL5 ; drug effects ; metabolism ; Diabetes Mellitus, Experimental ; drug therapy ; Diabetic Nephropathies ; drug therapy ; Diterpenes ; pharmacology ; Drugs, Chinese Herbal ; metabolism ; Epoxy Compounds ; pharmacology ; Glycated Hemoglobin A ; metabolism ; Immunosuppressive Agents ; pharmacology ; Kidney ; drug effects ; Kidney Diseases ; drug therapy ; Kidney Glomerulus ; metabolism ; Kidney Tubules ; metabolism ; Phenanthrenes ; pharmacology ; RNA, Messenger ; genetics ; Rats

AIM: To investigate the effect of DT-13 on gastric cancer cell migration, and to explore the possible mechanisms underlying the anti-metastasis activity of DT-13. METHODS: Growth inhibition of DT-13 was analyzed by the MTT assay. Cell migration was measured by the scratch-wound assay and transwell double chamber assay. To investigate the possible mechanisms underlying the anti-metastasis activity of DT-13, chemokine receptors that are involved in cancer metastasis (CCR2, CCR5, CCR7, CXCR4, and CXCR6) were detected by conventional PCR. The effect of DT-13 on CCR5 and CXCR4 expression was further evaluated by quantitative PCR and Western blot, respectively. The secretion of CCL5 (ligand of CCR5) and SDF-1 (ligand of CXCR4) were detected by enzyme-linked immunosorbent assay (ELISA). RESULTS: DT-13 inhibited BGC-823 and HGC-27 cell growth in a dose dependent manner, and the estimated IC50 value for 24 h treatment was 23.5 ± 5.1 μmol·L(-1) for BGC-823 cells and 35.6 ± 7.6 μmol·L(-1) for HGC-27 cells. DT-13 also significantly decreased gastric cancer cell migration. DT-13 significantly decreased the gene expression of CCR5 in both BGC-823 and HGC-27 gastric cancer cells, and moderately reduced the expression of CXCR4. Similar to the results of gene expression, significant down-regulation of CCR5 protein was observed, but CXCR4 protein levels were much less affected. CCL5 secretion, but not SDF-1 production, was inhibited by DT-13. CONCLUSION DT-13 inhibited gastric cancer cell migration by down-regulation of the CCR5-CCL5 axis.
Antineoplastic Agents, Phytogenic ; pharmacology ; Cell Movement ; drug effects ; Chemokine CCL5 ; analysis ; Down-Regulation ; Humans ; Neoplasm Metastasis ; drug therapy ; Receptors, CCR5 ; analysis ; Saponins ; pharmacology ; Stomach Neoplasms ; pathology ; Tumor Cells, Cultured

OBJECTIVETo investigate the effect of vitamin D on the expression of chemokine regulated on activation, normal T cells expressed and secreted (RANTES) in the lung tissue of asthmatic rats, and the role of vitamin D in the control of asthmatic airway inflammation and the synergistic action of hormones.

METHODSForty female Wistar rats were randomly and equally divided into normal control, asthma, vitamin D intervention, budesonide intervention, and budesonide+vitamin D intervention groups. Hematoxylin and eosin staining was used to observe pathological changes in the lung tissue. Immunohistochemistry was used to measure the protein expression of RANTES in lung tissue. Enzyme-linked immunosorbent assay was used to measure the level of RANTES in bronchoalveolar lavage fluid (BALF). Real-time quantitative PCR was used to measure the mRNA expression of RANTES.

RESULTSThe asthma group showed the most significant pathological changes in the lung tissue, including inflammatory cell infiltration, bronchial stenosis and distortion and smooth muscle rupture, while the intervention groups showed fewer pathological changes. Of the intervention groups, the budesonide intervention group showed fewer pathological changes than the vitamin D intervention group, and the budesonide+vitamin D intervention group showed the mildest pathological changes, which were similar to those observed in the normal control group. Protein expression of RANTES in the lung tissue and BALF was significantly higher in the asthma group than in the normal control group (P<0.05), while it was lower in the intervention groups than in the asthma group, exhibiting significant differences between each intervention group and the asthma group (P<0.05) (except the difference in protein expression of RANTES in BALF between the vitamin D intervention and asthma groups). The budesonide+vitamin D intervention group showed less protein expression of RANTES in the lung tissue and BALF than both the budesonide intervention and vitamin D intervention groups (P<0.05). The mRNA expression of RANTES was significantly higher in the asthma group than in the normal control group (P<0.05), while it was significantly lower in three intervention groups than in the asthma group (P<0.05), however no significant difference was found between the intervention groups in this regard. The budesonide+vitamin D intervention group showed the lowest level of RANTES mRNA, with no significant difference from the normal control group.

CONCLUSIONSThe mRNA and protein expression of RANTES in BALF and lung tissue increases significantly in asthmatic rats. Vitamin D intervention can decrease the expression of RANTES, suggesting that vitamin D can reduce airway inflammation by regulating the expression of RANTES. Vitamin D can be used together with budesonide to further decrease the mRNA and protein expression of RANTES.

Animals ; Asthma ; drug therapy ; metabolism ; Bronchoalveolar Lavage Fluid ; chemistry ; Budesonide ; therapeutic use ; Chemokine CCL5 ; analysis ; genetics ; Enzyme-Linked Immunosorbent Assay ; Female ; Immunohistochemistry ; Lung ; metabolism ; pathology ; Rats ; Rats, Wistar ; Vitamin D ; pharmacology