OBJECTIVE: To investigate the correlation of inducible co-stimulatory molecules (ICOS) with mesenteric vascular endothelial- mesenchymal transition (EndMT) and sclerosis in spontaneously hypertensive rats (SHR). METHODS: Twenty 4-week-old WKY rats and 20 SHRs of the same strain were both randomly divided into 4 groups for observation at 4, 6, 10 and 30 weeks of age. ICOS expression frequency in rat spleen CD4⁺T cells was analyzed using flow cytometry, and the expressions of ICOS, VE-cad, α-SMA and Col3 mRNA in rat mesentery were detected by RT-PCR. The distributions of ICOS, IL-17A and TGF-β in rat mesentery were detected by immunohistochemistry. The levels of IL-17A and TGF-β in rat plasma were measured using ELISA. The morphological changes of rat mesenteric vessels were observed with Masson staining. Spearman or Pearson correlation analyses were used to evaluate the correlation between ICOS expression and the expressions of the markers of vascular EndMT and sclerosis. RESULTS: Compared with the control WKY rats, the SHRs began to show significantly increased systolic blood pressure and ICOS expression frequency on CD4⁺T cells at 6 weeks of age (P < 0.05). In the SHRs, the mRNA and protein expressions of ICOS, α-SMA, Col3, IL-17A and TGF-β in the mesentery were significantly higher than those in control group (P < 0.05), while the mRNA and protein expressions of VE-cad started to reduce significantly at 10 weeks of age (P < 0.05). The plasma levels of IL-17A and TGF-β were significantly increased in SHRs since 6 weeks of age (P < 0.05) with progressive worsening of mesenteric vascular sclerosis (P < 0.05). ICOS mRNA and protein expression levels in the mesenteric tissues of SHRs began to show positive correlations with α-SMA and Col3 expression levels and the severity of vascular sclerosis at 6 weeks of age (P < 0.05) and a negative correlation with VE-cad expression level at 10 weeks (P < 0.05). CONCLUSION ICOS play an important pathogenic role in EndMT and sclerosis of mesenteric vessels in essential hypertension by mediating related immune responses.
Rats ; Animals ; Rats, Inbred SHR ; Rats, Inbred WKY ; Hypertension ; Interleukin-17 ; Sclerosis/pathology* ; Transforming Growth Factor beta ; Mesentery/pathology* ; RNA, Messenger/metabolism* ; Blood Pressure

OBJECTIVE: To investigate the effect and relative mechanism of Recombinant Human Thrombopoietin (rhTPO) on long-term hematopoietic recovery in mice with acute radiation sickness. METHODS: Mice were intramuscularly injected with rhTPO (100 μg/kg) 2 hours after total body irradiation with ⁶⁰Co γ-rays (6.5 Gy). Moreover, six months after irradiation, peripheral blood, hematopoietic stem cells (HSC) ratio, competitive transplantation survival rate and chimerization rate, senescence rate of c-kit⁺ HSC, and p16 and p38 mRNA expression of c-kit⁺ HSC were detected. RESULTS: Six months after 6.5 Gy γ-ray irradiation, there were no differences in peripheral blood white blood cells, red blood cells, platelets, neutrophils and bone marrow nucleated cells in normal group, irradiated group and rhTPO group (P>0.05). The proportion of hematopoietic stem cells and multipotent progenitor cells in mice of irradiated group was significantly decreased after irradiation (P<0.05), but there was no significant changes in rhTPO group (P>0.05). The counts of CFU-MK and BFU-E in irradiated group were significantly lower than that in normal group, and rhTPO group was higher than that of the irradiated group(P<0.05). The 70 day survival rate of recipient mice in normal group and rhTPO group was 100%, and all mice died in irradiation group. The senescence positive rates of c-kit⁺ HSC in normal group, irradiation group and rhTPO group were 6.11%, 9.54% and 6.01%, respectively (P<0.01). Compared with the normal group, the p16 and p38 mRNA expression of c-kit⁺ HSC in the irradiated mice were significantly increased (P<0.01), and it was markedly decreased after rhTPO administration (P<0.01). CONCLUSION The hematopoietic function of mice is still decreased 6 months after 6.5 Gy γ-ray irradiation, suggesting that there may be long-term damage. High-dose administration of rhTPO in the treatment of acute radiation sickness can reduce the senescence of HSC through p38-p16 pathway and improve the long-term damage of hematopoietic function in mice with acute radiation sickness.
Humans ; Mice ; Animals ; Thrombopoietin/metabolism* ; Hematopoietic Stem Cells ; Blood Platelets ; Recombinant Proteins/therapeutic use* ; Radiation Injuries ; RNA, Messenger/metabolism*

OBJECTIVE: To explore the role of interleukin-17A (IL-17A) in renal epithelial- mesenchymal transition (EMT) in essential hypertensive nephropathy. METHODS: Four-week-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (control group) were both randomized into 4 groups (n=5) for observation at 4, 6, 10 and 30 weeks of age. Blood pressure of the rats was monitored using a noninvasive tail artery blood pressure measurement instrument. The percentage of Th17 cells in the splenocytes was analyzed using flow cytometry. The mRNA and protein expression levels of IL-17A, iNOS, Arg-1, E-cadherin, and α-SMA in the kidneys of the rats were detected using RT-PCR and immunohistochemical staining, respectively, and plasma levels of IL-17A were regularly detected using ELISA. RESULTS: At the age of 6 weeks, the SHRs began to show significantly higher blood pressure with greater Th17 cell percentage in the splenocytes and high renal expression and plasma level of IL-17A than WKY rats (P < 0.05 or P < 0.01). At 30 weeks, renal expression of E-cadherin mRNA and protein was significantly lower and the expression of Arg-1 mRNA and protein was significantly higher in SHR than in WKY rats (P < 0.01). Compared with the WKY rats, the SHRs showed significantly higher mRNA and protein expressions of iNOS at 6 and 10 weeks (P < 0.05 or 0.01) and higher α-SMA mRNA and protein expressions since 10 weeks of age (P < 0.05 or 0.01). In SHRs older than 10 weeks, renal IL-17A mRNA and protein expression levels were negatively correlated with those of E-cadherin (r=-0.731, P < 0.05; r=-0.827, P < 0.01) and positively correlated with those of α-SMA (r=0.658, P < 0.05; r=0.968, P < 0.01). CONCLUSION IL-17A is closely correlated with the progression of renal EMT in SHR and plays its role possibly by mediating M1/M2 polarization of renal infiltrating macrophages.
Animals ; Blood Pressure ; Cadherins/metabolism* ; Epithelial-Mesenchymal Transition ; Hypertension ; Interleukin-17/metabolism* ; Kidney ; RNA, Messenger/metabolism* ; Rats ; Rats, Inbred SHR ; Rats, Inbred WKY

This study aims to investigate the efficacy of forsythiaside A(FTA) against CCl_4-induced liver fibrosis and the mechanism. Specifically, activities of serum alanine/aspartate aminotransferase(ALT/AST) and hydroxyproline(HYP) level in liver were detected, and pathological morphology of liver was observed based on hematoxylin-eosin(HE) staining, Masson's trichrome staining, and Sirius red staining of liver. On this basis, the effect of FTA on liver fibrosis was evaluated. The mRNA expression of actin alpha 2/α-smooth muscle actin(Acta2/α-SMA), transforming growth factor β(Tgfβ), collagen Ⅰ alpha 1(Col1 a1), and collagen Ⅲ alpha 1(Col3 a1) in liver tissue and hepatic stellate cells(HSC) was determined by qPCR, and the protein expression of α-SMA in liver tissue and HSC was measured by Western blot to assess the inhibition of FTA on HSC activation. The protein expression of α-SMA, vi-mentin(Vim), vascular endothelial cadherin(Ve-cadherin), and platelet endothelial cell adhesion molecule-1(PECAM-1/CD31) was measured by Western blot to evaluate the reverse of endothelial-mesenchymal transition(EMT) by FTA. The efficacy of FTA in relieving CCl_4-induced liver fibrosis was evidenced by the alleviation of hepatocyte necrosis, liver inflammation, and hepatic collagen deposition. FTA decreased the mRNA expression of Acta2, Tgfβ, Col1 a1, and Col3 a1 and protein expression of α-SMA both in vivo and in vitro. FTA reversed the increase of α-SMA and Vim and the decrease of CD31 and Ve-cadherin in livers from mice treated with CCl_4. Therefore, FTA alleviated CCl_4-induced liver fibrosis in mice via suppressing HSC activation and reversing EMT.
Animals ; Mice ; Actins/metabolism* ; Alanine Transaminase/blood* ; Carbon Tetrachloride/metabolism* ; Collagen/metabolism* ; Hepatic Stellate Cells ; Liver/drug effects* ; Liver Cirrhosis/genetics* ; RNA, Messenger/metabolism* ; Transforming Growth Factor beta/metabolism* ; Glycosides/therapeutic use*

OBJECTIVE: To determine the effects of hawthorn extract on serum lipid levels, pathological changes in aortic atherosclerosis plaque, inflammatory factors, and apoptosis-related protein and mRNA expression in apolipoprotein E gene knockout (ApoE) mice. METHODS: Thirty-six ApoE mice were fed with a high-fat diet starting at the age of 8 weeks. Mice were randomly divided into 3 groups by a random number table including model group, hawthorn extract group, and simvastatin group, 12 mice in each group. Twelve 8-week-old C57BL/6 mice were fed a basic diet and served as control. The mice in the control and model groups were administered 0.2 mL saline daily, the mice in the hawthorn extract and simvastatin groups were administered with 50 mg/kg hawthorn extract or 5 mg/kg simvastatin daily for 16 weeks. After 16 weeks, plasma lipids including total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C) and high-density lipoprotein cholesterol (HDL-C) were determined by an enzymatic assay. Aortic atherosclerotic lesions were observed by light microscopy, scanning and transmission electron microscopy, respectively. Plasma levels of monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), adiponectin (APN), and hypersensitive C-reactive protein (hs-CRP) were measured by enzyme-linked immunosorbent assay (ELISA). Protein and mRNA expressions of Bax and Bcl-2 in the aorta were assessed by Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR), respectively. RESULTS: Compared to the control group, the plasma levels of TC, TG and LDL-C were significantly increased and HDL-C were significantly decreased in the model group (P<0.01). Compared to the model group, treatment with hawthorn extract significantly decreased the plasma levels of TC, TG, and LDL-C and increased the plasma level of HDL-C in ApoE mice (P<0.01). The levels of MCP-1, IL-1ß, and hs-CRP in the model group were significantly increased and APN was significantly decreased compared with the control group (P<0.01). Compared to the model group, treatment with hawthorn extract decreased the levels of MCP-1, IL-1ß, and hs-CRP and increased the APN level (P<0.01). Compared to the control group, the protein and mRNA expression of Bax in the model group were significantly increased and the expression of Bcl-2 was significantly decreased (P<0.01). Hawthorn extract also reduced the protein and mRNA expression of Bax and increased the Bcl-2 expression in the aorta (P<0.01). CONCLUSION Hawthorn extract has anti-atherosclerosis and stabilizing unstable plaque effects. The mechanism may be related to the inflflammation and apoptosis signaling pathways.
Animals ; Aorta ; pathology ; ultrastructure ; Apoptosis ; drug effects ; Atherosclerosis ; blood ; complications ; drug therapy ; Crataegus ; chemistry ; Inflammation ; blood ; complications ; drug therapy ; Inflammation Mediators ; metabolism ; Lipids ; blood ; Male ; Mice, Inbred C57BL ; Plant Extracts ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; bcl-2-Associated X Protein ; metabolism

OBJECTIVETo observe the effect of norcantharidin (NCTD) on collagen-induced arthritis (CIA) rats.

METHODSSixty Sprague-Dawley(SD) rats were randomly divided into 6 groups (n=10): normal group, CIA model group(model group), NCTD low-dose group [1.35 mg/(kg•d)], NCTD middle-dose group [2.7 mg/(kg•d)], NCTD high-dose group [5.4 mg/(kg•d)] and methotrexate (MTX) group [1.8 mg/(kg/w)]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin (H&E) staining. The serum levels of interleukin (IL) 1β, IL-6, tumor necrosis factor (TNF)-α, vascular endothelial growth factor (VEGF), IL-17 and transform growth factor (TGF) β were detected by enzyme linked immunosorbent assay (ELISA). The mRNA expression of retinoid-related orphan nuclear receptorγt (RORγt) and forkhead box P3 (Foxp3) in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction.

RESULTSMTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats' ankle joints compared with the model group (P<0.05 or P<0.01). All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats (P<0.05). Only middle- and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats (P<0.05). However, NCTD has no effect on vascular endothelial growth factor (VEGF) level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group (P<0.05). The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group (P<0.05).

CONCLUSIONSNCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.

Animals ; Arthritis, Experimental ; blood ; drug therapy ; pathology ; Bridged Bicyclo Compounds, Heterocyclic ; pharmacology ; therapeutic use ; Cytokines ; blood ; Forkhead Transcription Factors ; metabolism ; Joints ; drug effects ; pathology ; Male ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effect and potential mechanisms of rutaecarpine (Rut) in a rat artery balloon-injury model.

METHODSThe intimal hyperplasia model was established by rubbing the endothelia with a balloon catheter in the common carotid artery (CCA) of rats. Fifty rats were randomly divided into five groups, ie. sham, model, Rut (25, 50 and 75 mg/kg) with 10 rats of each group. The rats were treated with or without Rut (25, 50, 75 mg/kg) by intragastric administration for 14 consecutive days following injury. The morphological changes of the intima were evaluated by hematoxylin-eosin staining. The expressions of proliferating cell nuclear antigen (PCNA) and smooth muscle (SM) α-actin in the ateries were assayed by immunohistochemical staining. The mRNA expressions of c-myc, extracellular signal-regulated kinase 2 (ERK2), MAPK phosphatase-1 (MKP-1) and endothelial nitric oxide synthase (eNOS) were determined by real-time reverse transcription-polymerase chain reaction. The protein expressions of MKP-1 and phosphorylated ERK2 (p-ERK2) were examined by Western blotting. The plasma contents of nitric oxide (NO) and cyclic guanosine 3',5'-monophosphate (cGMP) were also determined.

RESULTSCompared with the model group, Rut treatment significantly decreased intimal thickening and ameliorated endothelial injury (P<0.05 or P<0.01). The positive expression rate of PCNA was decreased, while the expression rate of SM α-actin obviously increased in the vascular wall after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01). Furthermore, the mRNA expressions of c-myc, ERK2 and PCNA were downregulated while the expressions of eNOS and MKP-1 were upregulated (P<0.05 or P<0.01). The protein expressions of MKP-1 and the phosphorylation of ERK2 were upregulated and downregulated after Rut (50 and 75 mg/kg) administration (P<0.05 or P<0.01), respectively. In addition, Rut dramatically reversed balloon injury-induced decrease of NO and cGMP in the plasma (P<0.05 or P<0.01).

CONCLUSIONRut could inhibit the balloon injury-induced carotid intimal hyperplasia in rats, possibly mediated by promotion of NO production and inhibiting ERK2 signal transduction pathways.

Actins ; metabolism ; Animals ; Carotid Arteries ; drug effects ; metabolism ; pathology ; Carotid Artery Injuries ; drug therapy ; genetics ; pathology ; Cyclic GMP ; blood ; Disease Models, Animal ; Gene Expression Regulation ; drug effects ; Hyperplasia ; Indole Alkaloids ; pharmacology ; therapeutic use ; Male ; Nitric Oxide ; blood ; Phosphorylation ; drug effects ; Proliferating Cell Nuclear Antigen ; metabolism ; Quinazolines ; pharmacology ; therapeutic use ; RNA, Messenger ; genetics ; metabolism ; Rats, Sprague-Dawley ; Tunica Intima ; drug effects ; pathology

Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, CK2β, VEGF, GCL, GST, and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.
Blood Vessels ; Caseins ; Epithelial Cells ; Gene Expression ; Glutamate-Cysteine Ligase ; Glutathione Transferase ; Hydrogen-Ion Concentration ; Phosphoprotein Phosphatases ; Polymerase Chain Reaction ; Reactive Oxygen Species ; Retinaldehyde ; Reverse Transcription ; RNA, Messenger ; Signal Transduction ; Toxoplasma ; Toxoplasmosis ; Vascular Endothelial Growth Factor A

Obesity is currently one of the most serious public health problems and it can lead to numerous metabolic diseases. Leucrose, d-glucopyranosyl-α-(1-5)-d-fructopyranose, is an isoform of sucrose and it is naturally found in pollen and honey. The aim of this study was to investigate the effect of leucrose on metabolic changes induced by a high-fat diet (HFD) that lead to obesity. C57BL/6 mice were fed a 60% HFD or a HFD with 25% (L25) or 50% (L50) of its total sucrose content replaced with leucrose for 12 weeks. Leucrose supplementation improved fasting blood glucose levels and hepatic triglyceride content. In addition, leucrose supplementation reduced mRNA levels of lipogenesis-related genes, including peroxisome proliferator-activated receptor γ, sterol regulatory element binding protein 1C, and fatty acid synthase in HFD mice. Conversely, mRNA levels of β oxidation-related genes, such as carnitine palmitoyltransferase 1A and acyl CoA oxidase, returned to control levels with leucrose supplementation. Taken together, these results demonstrated the therapeutic potential of leucrose to prevent metabolic abnormalities by mediating regulation of plasma glucose level and hepatic triglyceride accumulation.
Acyl-CoA Oxidase ; Animals ; Blood Glucose ; Carnitine O-Palmitoyltransferase ; Diet, High-Fat ; Fasting ; Honey ; Lipogenesis ; Liver ; Metabolic Diseases ; Mice ; Mice, Obese ; Negotiating ; Obesity ; Peroxisomes ; Pollen ; Public Health ; RNA, Messenger ; Sterol Regulatory Element Binding Protein 1 ; Sucrose ; Triglycerides

OBJECTIVE: To investigate the effects of sera from rats fed with tablets (HGT) on endoplasmic reticulum (ER) stress in a steatotic hepatocyte model of free fatty acids (FFAs)-induced nonalcoholic fatty liver disease (NAFLD) and explore the possible mechanism. METHODS: FFAs prepared by mixing oleic acid and palmitic acid at the ratio of 2:1. HepG2 cells were treated with the sera from rats fed with low-, moderate-or high-dose HGT (HGT sera) or sera of rats fed with fenofibrate (fenofibrate sera), followed by treatment with 1 mmol/L FFAs for 24 h to induce hepatic steatosis. Oil red O staining was used to observe the distribution of lipid droplets in the cells. The biochemical parameters including triglyceride (TG), lactated hydrogenase (LDH), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured using a commercial kit. The morphological changes of the ER in the cells were observed using transmission electron microscopy. The protein/mRNA expressions of ER stress-related signal molecules including GRP78, PERK, p-PERK, ATF6, ATF4, CASPASE-12, CHOP, XBP-1, PKC, and p-PKC-δ were detected using Western blotting and/or quantitative real-time PCR (qRT-PCR). The changes in the protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP were also detected in cells with transient transfection of PKC-δ siRNA for PKC-δ knockdown. RESULTS: Compared with the control cells, the cells treated with FFAs showed significantly increased levels of TG, AST, and ALT ( < 0.05). Compared with FFAs-treated cells, the cells pretreated with HGT sera or fenofibrate sera all showed significantly decreased TG, AST and ALT levels ( < 0.05), reduced accumulation of the lipid droplets ( < 0.05), and lowered protein or mRNA expression levels of GRP78, p-PERK, ATF6, ATF4, CHOP, CASPASE-12, XBP-1 and p-PKC-δ ( < 0.05). PKC-δ knockdown caused significantly reduced protein expressions of GRP78, p-PERK, CASPASE-12 and CHOP in the cells with FFA-induced hepatic steatosis ( < 0.001); treatment with high-dose HGT serum more significantly reduced the expressions of GRP78 ( < 0.001) and P-PERK ( < 0.01) in FFAs-induced cells with PKC-δ knockdown. CONCLUSIONS HGT serum can effectively prevent FFAs-induced steatosis in HepG2 cells by alleviating ER stress, in which PKC-δ may act as an important target.
Alanine Transaminase ; blood ; Animals ; Aspartate Aminotransferases ; blood ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Endoplasmic Reticulum ; ultrastructure ; Endoplasmic Reticulum Stress ; drug effects ; Fatty Acids, Nonesterified ; Fenofibrate ; administration & dosage ; Hep G2 Cells ; Humans ; Hypolipidemic Agents ; administration & dosage ; Microscopy, Electron, Transmission ; Non-alcoholic Fatty Liver Disease ; blood ; etiology ; prevention & control ; RNA, Messenger ; blood ; Rats ; Serum ; Tablets ; Triglycerides ; blood