In order to investigate whether the effect of Yuxuebi Tablets on the peripheral and central inflammation resolution of mice with inflammatory pain is related to their regulation of G protein-coupled receptor 37(GPR37), an inflammatory pain model was established by injecting complete Freund's adjuvant(CFA) into the paws of mice, with a sham-operated group receiving a similar volume of normal saline. The mice were assigned randomly to the sham-operated group, model group, ibuprofen group(91 mg·kg~(-1)), and low-, medium-, and high-dose groups of Yuxuebi Tablets(60, 120, and 240 mg·kg~(-1)). The drug was administered orally from days 1 to 19 after modeling. Von Frey method and the hot plate test were used to detect mechanical pain thresholds and heat hyperalgesia. The levels of interleukin-10(IL-10) and transforming growth factor-beta(TGF-β) in the spinal cord were quantified using enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein expression of GPR37 in the spinal cord was measured by real-time quantitative reverse transcription PCR(qRT-PCR) and Western blot. Additionally, immunofluorescence was used to detect the expression of macrosialin antigen(CD68), mannose receptor(MRC1 or CD206), and GPR37 in dorsal root ganglia, as well as the expression of calcium-binding adapter molecule 1(IBA1), CD206, and GPR37 in the dorsal horn of the spinal cord. The results showed that compared with those of the sham-operated group, the mechanical pain thresholds and hot withdrawal latency of the model group significantly declined, and the expression of CD68 in the dorsal root ganglia and the expression of IBA1 in the dorsal horn of the spinal cord significantly increased. The expression of CD206 and GPR37 significantly decreased in the dorsal root ganglion and dorsal horn of the spinal cord, and IL-10 and TGF-β levels in the spinal cord were significantly decreased. Compared with those of the model group, the mechanical pain thresholds and hot withdrawal latency of the high-dose group of Yuxuebi Tablets significantly increased, and the expression of CD68 in the dorsal root ganglion and IBA1 in the dorsal horn of the spinal cord significantly decreased. The expression of CD206 and GPR37 in the dorsal root ganglion and dorsal horn of the spinal cord significantly increased, as well as IL-10 and TGF-β levels in the spinal cord. These findings indicated that Yuxuebi Tablets may reduce macrophage(microglial) infiltration and foster M2 macrophage polarization by enhancing GPR37 expression in the dorsal root ganglia and dorsal horn of the spinal cord of CFA-induced mice, so as to improve IL-10 and TGF-β levels, promote resolution of both peripheral and central inflammation, and play analgesic effects.
Inflammation/genetics* ; Pain/genetics* ; Drugs, Chinese Herbal/administration & dosage* ; Animals ; Mice ; Freund's Adjuvant/pharmacology* ; Ibuprofen ; Pain Threshold/drug effects* ; Hyperalgesia/genetics* ; Ganglia, Spinal ; Interleukin-10/genetics* ; Transforming Growth Factor beta/genetics* ; Reverse Transcriptase Polymerase Chain Reaction ; Tablets ; Receptors, G-Protein-Coupled

This study aimed to investigate the potential mechanism and the compatibility significance of Tanyu Tongzhi Formula in treating atherosclerosis(AS) in mice based on the transforming growth factor-β(TGF-β)/Smad2/3 signaling pathway. Eight C57BL/6J mice were as assigned to a normal control group and fed a regular diet, while 35 ApoE~(-/-) mice of the same strain were fed a high-fat diet for 8 weeks to establish an AS model. The model mice were randomly divided into a model group, a Tanyu Tongzhi group(18.2 mg·kg~(-1)), a Huatan(phlegm-resolving) group(10.4 mg·kg~(-1)), and a Quyu(blood stasis-resolving) group(7.8 mg·kg~(-1)), with 8 mice in each group. Except for the normal group, all other groups continued to be fed a high-fat diet for 8 weeks to maintain the AS model, and then the mice were treated by gavage for 8 weeks. Plasma levels of total cholesterol(TC), triglycerides(TG), low-density lipoprotein cholesterol(LDL-C), high-density lipoprotein cholesterol(HDL-C), interleukin-1β(IL-1β), and interleukin-18(IL-18) were measured using enzyme-linked immunosorbent assay(ELISA). Hematoxylin and eosin(HE) staining, oil red O staining, and Russell-Movat pentachrome staining were performed to observe the pathological changes in the aortic tissue. The proportions of aortic plaque area, lipid-stained area, collagen fibers, and elastic fibers were calculated. Immunofluorescence was used to detect the protein expression levels of matrix metalloproteinase 2(MMP2) and tissue inhibitor of metalloproteinases 2(TIMP2). Western blot was used to detect the protein expression levels of TGF-β1, TGF-β2, Smad2/3, and Smad7 in aortic tissue. Real-time fluorescence quantitative PCR(RT-qPCR) was used to measure the mRNA expression levels of TGF-β receptor(TGF-βR), TGF-β1, Smad2/3, Smad7, intercellular adhesion molecule-1(ICAM-1), and vascular cell adhesion molecule-1(VCAM-1) in aortic tissue. The results showed that compared with the normal control group, the model group had increased plasma TC and LDL-C, significantly decreased HDL-C, and significantly elevated plasma IL-1β and IL-18 levels. The model group also exhibited an increased proportion of aortic plaque area, lipid-stained area, and collagen fiber area, along with significantly upregulated MMP2 and downregulated TIMP2 expression in the aortic arch. Additionally, the expression levels of TGF-βR, TGF-β1, and p-Smad2/3 proteins and mRNA in the aortic tissue were significantly elevated, while Smad7 expression was decreased. Compared with the model group, the Tanyu Tongzhi group showed significantly reduced plasma TC and LDL-C levels, significantly increased HDL-C levels, and significantly decreased plasma IL-1β and IL-18 levels. The Tanyu Tongzhi group also exhibited a significant reduction in aortic plaque size and severity, a significant downregulation of MMP2 expression in the aortic arch, and significantly decreased ICAM-1 and VCAM-1 mRNA expression levels. Moreover, the Tanyu Tongzhi group demonstrated significantly reduced expression levels of TGF-β1 and p-Smad2/3 proteins and mRNA in the aortic tissue, and an increased expression level of Smad7 protein to varying degrees. Compared with the Tanyu Tongzhi group, the Quyu group had significantly higher LDL-C levels and elevated plasma IL-1β and IL-18 levels. The Huatan group showed upregulated MMP2 expression and downregulated TIMP2 expression in the aortic arch. In conclusion, Tanyu Tongzhi Formula, which is composed based on the pathogenesis of phlegm and blood stasis, maintains vascular homeostasis by primarily regulating lipid metabolism and controlling inflammatory factors through the Huatan group, and maintaining vascular wall permeability, inhibiting plaque development, and stabilizing plaques through the Quyu group. The mechanism of action may involve inhibiting TGF-β1 expression in the aorta, reducing Smad2/3 phosphorylation, and simultaneously increasing Smad7 expression.
Animals ; Atherosclerosis/metabolism* ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Mice ; Mice, Inbred C57BL ; Male ; Transforming Growth Factor beta/genetics* ; Humans ; Homeostasis/drug effects* ; Aorta/metabolism* ; Smad2 Protein/genetics* ; Smad3 Protein/genetics*

OBJECTIVE: To evaluate the efficacy of rapmycin for treatment of experimental autoimmune encephalomyelitis (EAE) in mice and explore the underlying mechanism. METHODS: An EAE model was established in C57BL/6 mice. After immunization, the mice were divided into model group and rapamycin groups treated daily with low-dose (0.3 mg/kg) or high-dose (1 mg/kg) rapamycin. The clinical scores of the mice were observed using Knoz score, the infiltration of IL-17 cells in the central nervous system (CNS) was determined using immunohistochemistry; the differentiation of peripheral Treg cells was analyzed using flow cytometry, and the changes in the levels of cytokines were detected with ELISA; the changes in the expressions of p-Smad2 and p- smad3 were investigated using Western blotting. RESULTS: High-dose rapamycin significantly improved the neurological deficits scores of EAE mice. In high-dose rapamycin group, the scores in the onset stage, peak stage and remission stage were 0.14±0.38, 0.43±1.13 and 0.14±0.37, respectively, as compared with 1.14±0.69, 2.14±1.06 and 2.2±0.75 in the model group. The infiltration of inflammatory IL-17 cells was significantly lower in high-dose rapamycin group than in the model group (43±1.83 153.5±7.02). High-dose rapamycin obviously inhibited the production of IL-12, IFN-γ, IL-17 and IL-23 and induced the anti-inflammatory cytokines IL-10 and TGF-β. The percentage of Treg in CD4+ T cells was significantly higher in high- dose rapamycin group than in the model group (10.17 ± 0.68 3.52 ± 0.32). In the experiment, combined treatments of the lymphocytes isolated from the mice with rapamycin and TGF-β induced a significant increase in the number of Treg cells (13.66±1.89) compared with the treatment with rapamycin (6.23±0.80) or TGF-β (4.87±0.85) alone. Rapamycin also obviously up-regulated the expression of p-Smad2 and p-Smad3 in the lymphocytes. CONCLUSIONS Rapamycin can promote the differentiation of Treg cells by up-regulating the expression of p-Smad2 and p-smad3 to improve neurological deficits in mice with EAE.
Animals ; Anti-Inflammatory Agents ; administration & dosage ; therapeutic use ; Cell Differentiation ; drug effects ; Encephalomyelitis, Autoimmune, Experimental ; drug therapy ; metabolism ; Interferon-gamma ; metabolism ; Interleukins ; metabolism ; Lymphocytes ; cytology ; Mice ; Mice, Inbred C57BL ; Sirolimus ; administration & dosage ; therapeutic use ; Smad Proteins ; metabolism ; T-Lymphocytes, Regulatory ; cytology ; drug effects ; Transforming Growth Factor beta ; metabolism ; Up-Regulation

The traditional Chinese medicine Shensong Yangxin (SSYX) can improve the clinical symptoms of arrhythmia in an integrated manner. This study aimed to investigate the electrophysiological effect of SSYX on the hearts of myocardial-infarcted rabbits and further explore the mechanism by which SSYX alleviates myocardial fibrosis. Myocardial infarction (MI) was established in rabbits by ligation of the left circumflex coronary. The rabbits were treated with SSYX (0.5 g/kg/d) or saline for 8 weeks by oral administration. Microelectrode array (MEA) technology was used in vivo for extracellular electrophysiological recordings of the infarct border zone. Masson's trichrome staining was used to observe myocardial fibrosis. Western blotting was performed to evaluate the protein expression levels of collagen I (COL I) and collagen III (COL III). Quantitative real-time polymerase chain reaction (real-time PCR) was performed to evaluate the TGF-β1 and MMP-2 mRNA expression levels. The results showed that the total activation time (TAT) and the dispersion of TAT were significantly increased and the excitation propagation markedly disordered after MI. SSYX could significantly decrease TAT and the dispersion of TAT, and significantly ameliorate the chaotic spread pattern of excitation. Furthermore, SSYX treatment could significantly decrease COL I and COL III protein levels and down-regulate TGF-β1 and MMP-2 mRNA expression levels in MI rabbits. It was concluded that SSYX may ameliorate cardiac electrophysiological abnormalities in infarcted hearts by decreasing the protein levels of COL I and COL III, down-regulating the mRNA expression levels of TGF-β1 and MMP2, and thereby reducing adverse cardiac remodeling.
Animals ; Collagen Type I ; genetics ; metabolism ; Collagen Type III ; genetics ; metabolism ; Drugs, Chinese Herbal ; administration & dosage ; pharmacology ; therapeutic use ; Female ; Heart Rate ; drug effects ; Hyperplasia ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Myocardial Infarction ; drug therapy ; pathology ; Myocardium ; metabolism ; pathology ; Rabbits ; Transforming Growth Factor beta ; genetics ; metabolism

Renal tubulointerstitial fibrosis is the common ending of progressive renal disease. It is worth developing new ways to stop the progress of renal fibrosis. Peroxisome proliferator-activated receptor-γ (PPARγ) agonists have been studied to treat diabetic nephropathy, cisplatin-induced acute renal injury, ischemia reperfusion injury and adriamycin nephropathy. In this study, unilateral ureteral obstruction (UUO) was used to establish a different renal fibrosis model. PPAR? agonist pioglitazone was administrated by oral gavage and saline was used as control. At 7th and 14th day after the operation, mice were sacrificed for fibrosis test and T lymphocytes subsets test. Unexpectedly, through MASSON staining, immunohistochemistry for α-SMA, and Western blotting for a-SMA and PDGFR-β, we found that pioglitazone failed to attenuate renal fibrosis in UUO mice. However, flow cytometry showed that pioglitazone down-regulated Th1 cells, and up-regulated Th2 cells, Th17 cells and Treg cells. But the Th17/Treg ratio had no significant change by pioglitazone. Real-time PCR results showed that TGF-β and MCP-1 had no significant changes, at the same time, CD4(+) T cells associated cytokines were partially regulated by pioglitazone pretreatment. Taken together, pioglitazone failed to suppress renal fibrosis progression caused by UUO.
Animals ; Chemokine CCL2 ; metabolism ; Fibrosis ; Kidney ; pathology ; Kidney Diseases ; drug therapy ; etiology ; Male ; Mice ; Mice, Inbred C57BL ; PPAR gamma ; agonists ; T-Lymphocyte Subsets ; drug effects ; Thiazolidinediones ; administration & dosage ; pharmacology ; therapeutic use ; Transforming Growth Factor beta ; metabolism ; Urethral Obstruction ; complications

OBJECTIVETo study the effect of Fuzheng Sanjie recipe in regulating tumor-associated macrophages (TAMs) in Lewis lung cancer mice.

METHODEfforts were made to establish the Lewis lung cancer mouse model, weigh tumors and calculate the anti-tumor rate. The immunohistochemical method was used to examine the infiltration degree of CD68 + in tumor tissues in each group. ELISA was used to examine the content of IFN-γ, TGF-β, IL-4, IL-13, IL-6, IL-10, IL-12, TNF-α in mice serum.

RESULTCompared with the tumor-bearing model group, all of the other groups showed higher tumor inhibition rates, i. e. 50.28% for the DDP group, 34.37% for the TCM-preventing group and 66.76% for the Chinese and western medicine group, with statistical difference (P < 0.05), but without statistical difference in the infiltration degree of CD68+. The expressions of the IFN-γ, IL-6, IL-12 in tumor-bearing groups were lower than that in the blank control group, but with higher contents of IL-4, IL-13, TGF-&beta;. Intervened with different drugs, there were significant differences in content among some relevant cytokines (P < 0.05), as well as statistical differences among the TCM prevention group, the Chinese and western medicine group and the tumor-bearing control group (P <0. 05) , but without statistical difference in TNF-α and IL-10 content from the tumor-bearing control group (P < 0.05).

CONCLUSIONFuzheng Sanjie recipe could reverse the immune remodeling effect and control the tumor growth by down-regulating the expressions of IL-4, IL-13, TGF-α in lung cancer immune microenvironment and up-regulating the expression of IFN-γ.

Animals ; Cell Line, Tumor ; Disease Models, Animal ; Drugs, Chinese Herbal ; administration & dosage ; Humans ; Interleukin-10 ; blood ; Interleukin-12 ; blood ; Interleukin-13 ; blood ; Lung Neoplasms ; blood ; drug therapy ; immunology ; Macrophages ; drug effects ; immunology ; Male ; Mice ; Mice, Inbred C57BL ; Transforming Growth Factor beta ; blood ; Tumor Necrosis Factor-alpha ; blood

Oestrogen is essential for maintaining bone mass, and it has been demonstrated to induce osteoblast proliferation and bone formation. In this study, complementary DNA (cDNA) microarrays were used to identify and study the expression of novel genes that may be involved in MC3T3-E1 cells' response to 17-&beta; estradiol. MC3T3-E1 cells were inoculated in minimum essential media alpha (α-MEM) cell culture supplemented with 17-&beta; estradiol at different concentrations and for different time periods. MC3T3-E1 cells treated with 10⁻⁸ mol⋅L⁻¹ 17-&beta; estradiol for 5 days exhibited the highest proliferation and alkaline phosphatase (ALP) activity; thus, this group was chosen for microarray analysis. The harvested RNA was used for microarray hybridisation and subsequent real-time reverse transcription polymerase chain reaction (RT-PCR) to validate the expression levels for selected genes. The microarray results were analysed using both functional and pathway analysis. In this study, microarray analysis detected 5403 differentially expressed genes, of which 1996 genes were upregulated and 3407 genes were downregulated, 1553 different functional classifications were identified by gene ontology (GO) analysis and 53 different pathways were involved based on pathway analysis. Among the differentially expressed genes, a portion not previously reported to be associated with the osteoblast response to oestrogen was identified. These findings clearly demonstrate that the expression of genes related to osteoblast proliferation, cell differentiation, collagens and transforming growth factor beta (TGF-&beta;)-related cytokines increases, while the expression of genes related to apoptosis and osteoclast differentiation decreases, following the exposure of MC3T3-E1 cells to α-MEM supplemented with 17-&beta; estradiol. Microarray analysis with functional gene classification is critical for a complete understanding of complementary intracellular processes. This microarray analysis provides large-scale gene expression data that require further confirmatory studies.
3T3 Cells ; Alkaline Phosphatase ; drug effects ; Animals ; Apoptosis ; drug effects ; genetics ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; genetics ; Cell Proliferation ; drug effects ; Cell Survival ; drug effects ; genetics ; Collagen ; drug effects ; genetics ; Coloring Agents ; Cytokines ; drug effects ; genetics ; Estradiol ; administration & dosage ; pharmacology ; Estrogens ; administration & dosage ; pharmacology ; Gene Expression Profiling ; Mice ; Oligonucleotide Array Sequence Analysis ; Osteoblasts ; drug effects ; Signal Transduction ; drug effects ; genetics ; Tetrazolium Salts ; Thiazoles ; Transforming Growth Factor beta ; drug effects ; genetics

OBJECTIVETo explore the effects of Yifei Huoxue Granule (YFHXG) on the proliferation and the transforming growth factor-beta (TGF-beta) activity of rat pulmonary artery smooth muscle cells (PASMCs) induced by platelet-derived growth factor BB (PDGF-BB).

METHODSUsing tissue block adhering wall method, the primary rat PASMCs were cultured. PASMCs at the log phase growth were randomly divided into the control group, the PDGF-BB group, the PDGF-BB + high YFHXG group (at the final concentration of 7.5 mg/mL), the PDGF-BB + middle YFHXG group (at the final concentration of 1.5 mg/mL), and the PDGF-BB + low YFHXG group (at the final concentration of 0.3 mg/mL), respectively. MTT assay were employed to determine the cell proliferation rate of each group. Flow cytometric analyses were used to detect the cell cycle constituent ratio and the proliferation index (PI). In addition, TGF-beta protein's expression was determined by immunocytochemical assay (SP method).

RESULTSCompared with the control group, the proliferation of PASMCs in the PDGF-BB group was obviously active (P<0.01). But when compared with the PDGF-BB group, along with the increased concentration of YFHXG, the growth of PASMCs was obviously inhibited, the cell ratio of G0/G1, phase obviously increased, the cell ratio of S + G2/M phase significantly decreased, and PI significantly decreased. Besides, the expression of TGF-beta protein decreased in a dose-dependent manner (P<0.05, P<0.01).

CONCLUSIONSPDGF-BB could directly stimulate the proliferation of PASMCs. YFHXG had a significant inhibition on the proliferation of rat PASMCs induced by PDGF-BB and could regulate the expression of TGF-beta.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drugs, Chinese Herbal ; pharmacology ; Female ; Male ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Myocytes, Smooth Muscle ; drug effects ; metabolism ; Proto-Oncogene Proteins c-sis ; administration & dosage ; pharmacology ; Pulmonary Artery ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta ; metabolism

BACKGROUNDCapsular contracture has become the most common complication associated with breast implant. Transforming growth factor-beta (TGF-&beta;) is well known for a prominent role in fibrotic diseases. Due to the critical role of TGF-&beta; in pathogenesis of capsular formation, we utilized thermosensitive C/GP hydrogel to controlled release of TGF-&beta; receptor kinase inhibitor (SD208) and investigated their effects on capsular contracture.

METHODSIn vitro degradation and drug release of C/GP hydrogel were performed. Twenty-four rabbits underwent subpanniculus implantation with 30 ml smooth silicone implants and were randomly divided into four groups as follows: Group 1 received saline solution; Group 2 received SD208; Group 3 received SD208-C/GP; Group 4 received C/GP. At 8 weeks, the samples of capsular tissues were analyzed by hematoxylin and eosin and immunohistological staining. The mRNA expression of collagen III and TGF-&beta;1 was detected by RT-PCR assay.

RESULTSC/GP hydrogel could be applied as an ideal drug delivery vehicle which supported the controlled release of SD208. SD208-C/GP treatment showed a significant reduction in capsule thickness with fewer vessels. The histological findings confirmed that the lower amounts of inflammatory cells and fibroblasts infiltrate in SD208-C/GP group. In contrast, typical capsules with more vessel predominance were developed in control group. We did not observe the same inhibitory effect of SD208 or C/GP treatment on capsular contracture. Moreover, SD208-C/GP therapy yielded an evident down-regulation of collagen III and TGF-&beta;1 mRNA expression.

CONCLUSIONSThis study demonstrated that controlled release of TGF-&beta; receptor kinase inhibitor from thermosensitive C/GP hydrogel could significantly prevent capsule formation after mammary implants.

Animals ; Breast Implantation ; adverse effects ; Chitosan ; chemistry ; Glycerophosphates ; chemistry ; Hydrogel, Polyethylene Glycol Dimethacrylate ; chemistry ; Immunohistochemistry ; Protein Kinase Inhibitors ; administration & dosage ; therapeutic use ; Rabbits ; Receptors, Transforming Growth Factor beta ; antagonists & inhibitors ; Reverse Transcriptase Polymerase Chain Reaction

Although 4,4'-diaminodiphenylsulfone (DDS, dapsone) has been used to treat several dermatologic conditions, including Hansen disease, for the past several decades, its mode of action has remained a topic of debate. We recently reported that DDS treatment significantly extends the lifespan of the nematode C. elegans by decreasing the generation of reactive oxygen species. Additionally, in in vitro experiments using non-phagocytic human fibroblasts, we found that DDS effectively counteracted the toxicity of paraquat (PQ). In the present study, we extended our work to test the protective effect of DDS against PQ in vivo using a mouse lung injury model. Oral administration of DDS to mice significantly attenuated the lung tissue damage caused by subsequent administration of PQ. Moreover, DDS reduced the local expression of mRNA transcripts encoding inflammation-related molecules, including endothelin-1 (ET-1), macrophage inflammatory protein-1alpha (MIP-1alpha), and transforming growth factor-beta (TGF-beta). In addition, DDS decreased the PQ-induced expression of NADPH oxidase mRNA and activation of protein kinase Cmicro (PKCmicro). DDS treatment also decreased the PQ-induced generation of superoxide anions in mouse lung fibroblasts. Taken together, these data suggest the novel efficacy of DDS as an effective protective agent against oxidative stress-induced tissue damages.
Animals ; Cells, Cultured ; Chemokine CCL3/drug effects/metabolism ; Dapsone/*administration & dosage ; Endothelin-1/drug effects/metabolism ; Fibroblasts/drug effects ; Herbicides/*antagonists & inhibitors/toxicity ; Lung Injury/chemically induced/*prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Oxidative Stress ; Paraquat/*antagonists & inhibitors/toxicity ; Protective Agents/*administration & dosage ; Protein Kinase C/genetics/metabolism ; Superoxides/analysis ; Transforming Growth Factor beta/drug effects/metabolism