This study aims to investigate the intervention effect of the aqueous extract of Epimedium sagittatum Maxim on the mouse model of bleomycin(BLM)-induced pulmonary fibrosis, so as to provide data support for the clinical treatment of pulmonary fibrosis. Ninety male C57BL/6N mice were randomized into normal(n=10), model(BLM, n=20), pirfenidone(PFD, 270 mg·kg~(-1), n=15), and low-, medium-, and high-dose E. sagittatum extract(1.67 g·kg~(-1), n=15; 3.33 g·kg~(-1), n=15; 6.67 g·kg~(-1), n=15) groups. The model of pulmonary fibrosis was established by intratracheal instillation of BLM(5 mg·kg~(-1)) in the other five groups except the normal group, which was treated with an equal amount of normal saline. On the day following the modeling, each group was treated with the corresponding drug by gavage for 21 days. During this period, the survival rate of the mice was counted. After gavage, the lung index was calculated, and the morphology and collagen deposition of the lung tissue were observed by hematoxylin-eosin(HE) and Masson staining, respectively. The levels of reactive oxygen species(ROS) in lung cell suspensions were measured by flow cytometry. The levels of glutathione peroxidase(GSH-Px), total superoxide dismutase(T-SOD), and malondialdehyde(MDA) the in lung tissue were measured. Terminal-deoxynucleoitidyl transferase-mediated nick-end labeling(TUNEL) was employed to examine the apoptosis of lung tissue cells. The content of interleukin-6(IL-6), chemokine C-C motif ligand 2(CCL-2), matrix metalloproteinase-8(MMP-8), transforming growth factor-beta 1(TGF-β1), alpha-smooth muscle actin(α-SMA), E-cadherin, collagen Ⅰ, and fibronectin in the lung tissue was measured by enzyme-linked immunosorbent assay(ELISA). The expression levels of F4/80, Ly-6G, TGF-β1, and collagen Ⅰ in the lung tissue were determined by immunohistochemistry. The mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue were determined by qRT-PCR. The content of hydroxyproline(HYP) in the lung tissue was determined by alkaline hydrolysation. The expression of α-SMA and E-cadherin was detected by immunofluorescence, and the protein levels of α-SMA, vimentin, E-cadherin in the lung tissue were determined by Western blot. The results showed the aqueous extract of E. sagittatum increased the survival rate, decreased the lung index, alleviated the pathological injury, collagen deposition, and oxidative stress in the lung tissue, and reduced the apoptotic cells. Furthermore, the aqueous extract of E. sagittatum down-regulated the protein levels of F4/80 and Ly-6G and the mRNA levels of CCL-2, IL-6, and MMP-7 in the lung tissue, reduced the content of IL-6, CCL-2, and MMP-8 in the alveolar lavage fluid. In addition, it lowered the levels of HYP, TGF-β1, α-SMA, collagen Ⅰ, fibronectin, and vimentin, and elevated the levels of E-cadherin in the lung tissue. The aqueous extract of E. sagittatum can inhibit collagen deposition, alleviate oxidative stress, and reduce inflammatory response by regulating the expression of the molecules associated with epithelial-mesenchymal transition, thus alleviating the symptoms of bleomycin-induced pulmonary fibrosis in mice.
Mice ; Male ; Animals ; Pulmonary Fibrosis/metabolism* ; Transforming Growth Factor beta1/metabolism* ; Epimedium/metabolism* ; Fibronectins/metabolism* ; Matrix Metalloproteinase 7/therapeutic use* ; Matrix Metalloproteinase 8/therapeutic use* ; Vimentin/metabolism* ; Interleukin-6/metabolism* ; Mice, Inbred C57BL ; Lung ; Collagen/metabolism* ; Bleomycin/toxicity* ; RNA, Messenger/metabolism* ; Cadherins/metabolism*

To study the effect of modified Buyang Huanwu Decoction on the hemorrhagic transformation after intravenous thrombolysis of recombinant tissue type plasminogen activator(rt-PA) in patients with super early(onset time<4. 5 h) cerebral infarction. From March 2016 to July 2018,at the brain disease zone of the First Affiliated Hospital of Henan University of Traditional Chinese Medicine,212 cases of super early cerebral infarction were selected and divided into two group according to the randomized complete blocks designs: control group(106 cases) and traditional Chinese medicine group(106 cases). The control group was treated with rt-PA intravenous thrombolysis,while the traditional Chinese medicine group was treated with modified Buyang Huanwu Decoction in addition to the therapy of the control group. Both groups were treated for 14 days. Neurological deficit score,serum matrix metalloproteinase-9(MMP-9),neuron specific enolase(NSE),vascular endothelial growth factor(VEGF) and plasma cellular fibronectin(c-FN) levels,the incidence of hemorrhagic transformation,clinical efficacy and adverse drug reactions before and after treatment were compared between the two groups. According to the findings,at the 14 thday after treatment,the rank sum test of the grade data showed that the clinical efficacy of the traditional Chinese medicine group was better than that of the control group(Z =-2. 033,P = 0. 042); on the basis of χ2 test,the total efficiency of the traditional Chinese medicine group was higher than that of the control group(χ2= 4. 895,P =0. 027); the hemorrhagic transformation rate of the traditional Chinese medicine group was lower than that of the control group within14 days of treatment(χ2= 3. 962,P = 0. 047). MMP-9 levels in the traditional Chinese medicine group were lower than those in the control group at the 3 rd,5 th,7 th,10 th,14 thd after treatment(t =-2. 474,-3. 022,-5. 163,-6. 998,-9. 821; P = 0. 014,0. 003,0,0,0). The improvement of c-FN,NSE,VEGF and NIHSS scores in the traditional Chinese medicine group was superior to that of the control group after 14 days of treatment(t =-2. 343,-3. 187,-2. 129,-3. 105; P = 0. 020,0. 002,0. 034,0. 002). No obvious adverse reactions of modified Buyang Huanwu Decoction were observed during 14 days of treatment. Modified Buyang Huanwu Decoction could reduce the expressions of MMP-9,c-FN,NSE and VEGF after rt-PA intravenous thrombolysis in patients with super early cerebral infarction,and decrease the hemorrhagic transformation rate after thrombolysis,with high safety.
Cerebral Infarction ; drug therapy ; Drugs, Chinese Herbal ; therapeutic use ; Fibronectins ; blood ; Humans ; Matrix Metalloproteinase 9 ; blood ; Medicine, Chinese Traditional ; Phosphopyruvate Hydratase ; blood ; Recombinant Proteins ; therapeutic use ; Thrombolytic Therapy ; Tissue Plasminogen Activator ; therapeutic use ; Vascular Endothelial Growth Factor A ; blood

OBJECTIVETo observe the anti-renal fibrosis effect of Paidu Baoshen Pill (PBP) on 5/6 nephrectomized rats and to explore its mechanism.

METHODSTotally 50 SD male healthy rats were randomly divided into the normal control group (n = 10), the sham-operation group (n = 10), and the nephrectomy model group (n = 30) according to the proportion of 1:1:3. Rats in the sham-operation group had their renal capsule isolated without nephrectomy. Rats in the nephrectomy model group had their kidneys 5/6 nephrectomized. Then 24 h urine was collected and 24 h urinary protein (24 h UP) detected. Serum blood urea nitrogen (BUN) and serum creatitine (SCr) were also tested. According to the SCr level 30 rats of the model group were further randomly divided into the model group, the PBP group, and the Niaoduqing Granule (NG) group, 10 in each group. Rats in the PBP group and the NG group were respectively administered with PBP (at the daily dose of 1.0 g/kg) and NG (at the daily dose of 3.33 g/kg) by gastrogavage (they were dissolved in distilled water). At the same time, 2 mL distilled water was administered by gastrogavage to rats in the normal control group, the sham-operation group, and the nephrectomy model group, once daily for 4 successive weeks. Mental conditions, activities, hair color, shape of stool, and the body weight were observed during administration. After 4 weeks, urine was collected to detect 24 h UP. Blood was sampled to detect SCr, BUN, transforming growth factor β1 (TGF-β1), type III procollagen (PC III), collagen type IV (Col IV), laminin (LN), and fibronectin (FN). After rats were killed, their left remnant renal tissues were collected for pathological examinations. The protein expression quantity of TGF-β1 and FN was detected by immunohistochemical method. mRNA expression levels of TGF-β1 and FN were detected using real time fluorescent quantitative PCR.

RESULTSThere was no statistical difference in the above indices between the normal control group and the sham-operation group (P > 0.05). Compared with the sham-operation group, rats' general condition was poorer in the model group, their body weight grew slower, and 24 h UP increased; serum levels of BUN, SCr, TGF-β1, PC III, Col IV, LN, and FN increased; the residual renal pathological lesion was serious; expression levels of TGF-β1, TGF-β1, mRNA, FN, and FN mRNA increased in the renal tissue (all P < 0.01). Compared with the model group, rats' general condition was better, their body weight grew faster, 24 h UP reduced (P < 0.05), blood levels of BUN and SCr decreased significantly (P < 0.01), serum levels of TGF-β1, PC III, CoL IV, LN, and FN decreased (P < 0.05, P < 0.01); the residual renal pathological lesion was attenuated in the PBP group and the NG group; expression levels of TGF-β1, TGF-β1, mRNA, FN, and FN mRNA decreased (P < 0.01). Compared with the NG group, blood levels of SCr and FN, and expression levels of FN and FN mRNA decreased more in the PBP group (P < 0.05).

CONCLUSIONSPBP had the effect of anti-renal fibro- sis in 5/6 nephrectomized rats. Down-regulating expression levels of TGF-β1, and FN from gene transcription and protein translation levels might be one of its mechanisms.

Animals ; Blood Urea Nitrogen ; Collagen Type IV ; Drugs, Chinese Herbal ; therapeutic use ; Fibronectins ; Kidney ; Kidney Diseases ; drug therapy ; Laminin ; Male ; Nephrectomy ; Rats ; Transforming Growth Factor beta1

The Goto-Kakizaki (GK) rat is a spontaneous type 2 diabetic animal model, which is characterized by a progressive loss of beta islet cells with fibrosis. In the present study, the hypoglycemic effect of asiatic acid (AA) in GK rats was examined. GK rats receiving AA at a daily dose of 25 mg·kg(-1) for four weeks showed a significant reduction in blood glucose levels. Age-matched normal Wistar rats were given 0.5% sodium carboxymethyl cellulose (CMC-Na) solution for the same periods and used as control. Compared to the normal Wistar rats, GK rats treated with AA showed improvement in insulin resistance partially through decreasing glucose level (P < 0.01) and insulin level (P < 0.05). Furthermore, the results of immunohistochemistry indicate that AA treatment reduced islet fibrosis in GK rats. Fibronectin, a key protein related to islet fibrosis, was over-expressed in GK rats, which was reversed significantly by AA treatment (P < 0.05). These findings suggest that AA has a beneficial effect on lowering blood glucose levels in GK rats and improves fibrosis of islets in diabetes, which may play a role in the prevention of islets dysfunction.
Animals ; Blood Glucose ; metabolism ; Centella ; chemistry ; Diabetes Mellitus, Type 2 ; drug therapy ; pathology ; Disease Models, Animal ; Fibronectins ; metabolism ; Fibrosis ; Glucose Tolerance Test ; Hyperglycemia ; drug therapy ; pathology ; Insulin ; blood ; Insulin Resistance ; Islets of Langerhans ; drug effects ; pathology ; Male ; Pancreatic Diseases ; metabolism ; pathology ; prevention & control ; Pentacyclic Triterpenes ; pharmacology ; therapeutic use ; Phytotherapy ; Plant Extracts ; pharmacology ; therapeutic use ; Rats, Inbred Strains

PURPOSE: This study was undertaken to investigate the effects of gamma linolenic acid (GLA) on inflammation and extracellular matrix (ECM) synthesis in mesangial and tubular epithelial cells under diabetic conditions. MATERIALS AND METHODS: Sprague-Dawley rats were intraperitoneally injected with either a diluent [n=16, control (C)] or streptozotocin [n=16, diabetes (DM)], and eight rats each from the control and diabetic groups were treated with evening primrose oil by gavage for three months. Rat mesangial cells and NRK-52E cells were exposed to medium containing 5.6 mM glucose and 30 mM glucose (HG), with or without GLA (10 or 100 microM). Intercellular adhesion molecule-1 (ICAM-1), monocyte chemoattractant protein-1 (MCP-1), and fibronectin (FN) mRNA and protein expression levels were evaluated. RESULTS: Twenty-four-hour urinary albumin excretion was significantly increased in DM compared to C rats, and GLA treatment significantly reduced albuminuria in DM rats. ICAM-1, MCP-1, FN mRNA and protein expression levels were significantly higher in DM than in C kidneys, and these increases were significantly abrogated by GLA treatment. In vitro, GLA significantly inhibited increases in MCP-1 mRNA expression and protein levels under high glucose conditions in HG-stimulated mesangial and tubular epithelial cells (p<0.05, respectively). ICAM-1 and FN expression showed a similar pattern to the expression of MCP-1. CONCLUSION: GLA attenuates not only inflammation by inhibiting enhanced MCP-1 and ICAM-1 expression, but also ECM accumulation in diabetic nephropathy.
Animals ; Anti-Inflammatory Agents/*therapeutic use ; Blotting, Western ; Chemokine CCL2/genetics/metabolism ; Diabetic Nephropathies/*drug therapy/*metabolism ; Enzyme-Linked Immunosorbent Assay ; Fibronectins/genetics/metabolism ; Intercellular Adhesion Molecule-1/genetics/metabolism ; Rats ; Rats, Sprague-Dawley ; Real-Time Polymerase Chain Reaction ; alpha-Linolenic Acid/*therapeutic use

OBJECTIVETo investigate the antifibrotic effect of the Chinese herbs Modified Danggui Buxue Decoction (, MDBD) on adraimycin-induced nephropathy in rats.

METHODSThirty-two male Sprague Dawley albino rats were randomly divided into 4 groups: the control, model, and two treatment groups, with 8 in each group. Nephropathy was induced in the latter 3 groups by intravenous injection of adriamycin. Rats in the two treatment groups received intragastric administration of benazepri (a positive control) or MDBD, which is composed of extracts of Radix Angelicae sinensis, Astragalus membranaceus (Fisch.) Bge and Rhizoma chuanxiong. Serum albumin, blood lipids, 24-h urine protein and urine N-acetyl-b-D-glucosaminidase (NAG) were measured every 2 weeks. The ratio of kidney to body weight was measured. The expressions of extracellular matrix proteins in the renal cortex, including colleagen IV (Col-IV) and fibronectin (FN), were examined by immunohistochemistry, and the transcription of genes encoding transforming growth factor β1 (TGF-β1), the tissue inhibitors of matrix metalloproteinase 1 (TIMP-1) and matrix metalloproteinase 9 (MMP-9) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) at the end of the 8-week treatment.

RESULTSCompared with the untreated rats in the model group, MDBD significantly increased serum albumin, lowered the blood lipids and decreased the ratio of kidney to body weight. MDBD significantly reduced the excretion levels of urinary protein and NAG as well as the accumulation of extracellular matrix (ECM), including Col-IV and FN, in the renal cortex. Further, MDBD decreased TIMP-1 and TGF-β1 gene expressions and increased MMP-9 gene expression in the kidney.

CONCLUSIONSMDBD was effective in treating the rat model of nephropathy. The clinical benefit was associated with reduction of renal fibrosis. The antifibrotic effect of MDBD may be mediated through the regulation of TIMP-1, MMP and TGF-β1 gene expressions.

Acetylglucosaminidase ; urine ; Animals ; Body Weight ; drug effects ; Collagen Type IV ; metabolism ; Doxorubicin ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Fibronectins ; metabolism ; Fibrosis ; Gene Expression Regulation ; drug effects ; Immunohistochemistry ; Kidney Cortex ; drug effects ; enzymology ; pathology ; physiopathology ; Kidney Diseases ; blood ; drug therapy ; physiopathology ; urine ; Kidney Function Tests ; Male ; Matrix Metalloproteinase 9 ; genetics ; metabolism ; Organ Size ; drug effects ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Serum Albumin ; metabolism ; Time Factors ; Tissue Inhibitor of Metalloproteinase-1 ; genetics ; metabolism ; Tissue Inhibitor of Metalloproteinase-2 ; genetics ; metabolism ; Transforming Growth Factor beta1 ; genetics ; metabolism ; Triglycerides ; blood

BACKGROUNDIn addition to hematopoietic effect, the erythropoietin is known as a multifunctional cytokine with anti-fibrosis and organ-protective activities. The purpose of this study was to evaluate the effect of recombinant human erythropoietin (rhEPO) on hepatic fibrosis and hepatic stellate cells (HSCs).

METHODSCarbon tetrachloride (CCl(4)) induced hepatic fibrosis mice models were used for in vivo study and HSCs line for in vitro study. CCl(4) and rhEPO (0, 200 or 1000 U/kg) was injected intraperitoneally in BALB/c mice three times a week for 4 weeks. Immunohistochemistry and immunoblotting were performed to evaluate expressions of transforming growth factor-β1 (TGF-β1), α-smooth muscle actin (α-SMA), and fibronectin in explanted liver. Immunoblotting of α-SMA, phophorylated Smad-2 and Smad-2/3 was performed in HSCs treated with TGF-β1 and/or rhEPO.

RESULTSExpressions of TGF-β1, α-SMA, and fibronectin were increased in CCl(4) injected mice livers, but significantly attenuated by co-treatment with CCl(4) and rhEPO. Co-treatment of rhEPO markedly suppressed fibrosis in Masson's trichrome compared with treatment of only CCl(4). TGF-β1 increased phosphorylated α-SMA, Smad-2 expressions in HSCs, which were decreased by rhEPO co-treatment.

CONCLUSIONSTreatment of rhEPO effectively suppressed fibrosis in CCl(4)-induced liver fibrosis mice models. Anti-fibrosis effect of rhEPO could be related to inhibition of TGF-β1 induced activation of HSCs.

Animals ; Carbon Tetrachloride ; toxicity ; Cells, Cultured ; Erythropoietin ; pharmacology ; therapeutic use ; Fibronectins ; analysis ; Hepatic Stellate Cells ; drug effects ; Liver Cirrhosis, Experimental ; metabolism ; prevention & control ; Male ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; pharmacology ; Smad2 Protein ; metabolism ; Transforming Growth Factor beta ; antagonists & inhibitors ; physiology

This study was aimed to prepare the polypeptide of N-terminal heparin-binding domain of fibronectin(rhFNHN-29 polypeptide) with pichia expression system, to detect biological activity of recombinant polypeptide and investigate its effect on disseminated intravascular coagulation (DIC) in rats. The sequence of N-terminal heparin-binding domain of fibronectin was amplified from FNcDNA by PCR. The aim gene was cloned into T vector for selection. Then it was cloned into pAo815SM and pPIC9K vectors.Lined pPIC9K vectors were transformed into GS115 Pichia cells so as to express the aim polypeptide in Pichia expression system. The fermentation liquid were precipitated by 80% ammonium sulfate, and the further dissolved sediment were purified using S-100 column and SP column. Its activity of binding with heparin were detected by Western-blot. The established DIC rats (40 rats) were randomly divided into two groups. One group was treated with rhFNHN-29 polypeptide, and the other was treated with normal saline. The rats in the former group were injected with rhFNHN-29 polypeptide (10 mg/kg) through tail vein at 0.5 hour before, 2 hours and 4 hours after injection of LPS respectively. The rats in latter group were injected with equal volume saline. In addition, 20 normal rats injected with normal saline were as normal controls. 500 microl blood was taken from the rat vein, at 6 hours after the injection of LPS. White blood cell (WBC), hemoglobin (Hb) and platelets were tested from 50 microl blood. The rest 450 microl blood was used to isolate plasma for detecting TNFa level and coagulogram. The rats were killed at 24 hours after injection with LPS. Their livers, lungs, hearts, kidneys, and brain tissues were taken for histopathologic examination. The results showed that the aim polypeptide was successfully expressed in Pichia expression system. The expression level reached approximately 30 mg/L. The polypeptide had activity of binding with heparin antibody. In the experiment study of polypeptide effect on DIC in rats, the plasma TNFa level in polypeptide-treated group was lower than that in saline control group, the hemogram, coagulogram and histopathology were more obviously improved in polypeptide-treated group as compared with saline control group. It is concluded that the rhFNHN-29 polypeptide is successfully prepared, this polypeptide can antagonize DIC induced by endotoxin in rats.
Animals ; Disseminated Intravascular Coagulation ; therapy ; Endotoxins ; Female ; Fibronectins ; genetics ; immunology ; therapeutic use ; Heparin ; metabolism ; Male ; Peptides ; genetics ; therapeutic use ; Pichia ; metabolism ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo observe the clinical effect of Busui Shengxue Granule (BSG) in treating chronic aplastic anemia (CAA) and the changes of serum fibronectin (Fn) level and expression of FMS-like tyrosine kinase 3 ligand (FL).

METHODSSixty-eight patients with CAA were assigned to two groups. The 35 patients in the test group were treated with BSG and the 33 in the control group by Zaizhang Shengxue Tablet. Levels of Fn and FL were determined before and after 6-month treatment by enzyme linked immunosorbent assay.

RESULTSThe clinical effective rate in the test group was 74.3% (26/35 cases), and that in the control group 48.5% (16/33 cases), the difference between the two groups was significant (P <0.05). In the test group, patients of yang-deficiency type showed an effect superior to that of yin-deficiency type, 93.8% (15/16 cases) vs 57.9% (11/19 cases, P < 0.05). High expression manners of Fn and FL were shown in all CAA patients, the high expressions of both were lowered to some extent after treatment, but the improvement in the test group was better than that in the control group (P <0.05), and the deviation was more liable to be corrected in patients of Shen-yang deficiency type than that in patients of Shen-yin deficiency type.

CONCLUSIONBSG is better than Zaizhang Shengxue Tablet in clinical effect and improving the CAA related adhesive molecules, Fn and FL, suggesting that it is an effective remedy for preventing and treating CAA, with its curative effect more significant on patients of yang-deficiency type than that on patients of yin-deficiency type.

Adolescent ; Adult ; Aged ; Anemia, Aplastic ; blood ; drug therapy ; Child ; Child, Preschool ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Fibronectins ; blood ; Humans ; Male ; Middle Aged ; Young Adult ; fms-Like Tyrosine Kinase 3 ; antagonists & inhibitors

OBJECTIVETo investigate the effects of Tongluo Recipe on the expression of collagen IV (Col IV), fibronectin (FN), laminin (LN), transforming growth factor-beta1 (TGF-beta1) in rat renal tissues and explore the mechanism underlying these effects in rats with glomerular sclerosis.

METHODSThe pathological changes in the renal tissues of rats with glomerular sclerosis were observed microscopically, and the expressions of Col IV, FN, LN, and TGF-beta1 were detected using immunohistochemical staining and image analysis system.

RESULTSTongluo Recipe significantly decreased the expressions of Col IV, FN, LN and TGF-beta1 in the renal tissue of rats with glomerular sclerosis (P<0.05 or P<0.01) and obviously alleviated the renal pathologies (P<0.01).

CONCLUSIONThe therapeutic effects of Tongluo Recipe are probably mediated by lowered expressions of Col IV, FN, LN and TGF-beta1.

Animals ; Collagen Type IV ; biosynthesis ; Drugs, Chinese Herbal ; therapeutic use ; Fibronectins ; biosynthesis ; Glomerulosclerosis, Focal Segmental ; drug therapy ; metabolism ; pathology ; Kidney ; drug effects ; metabolism ; pathology ; Male ; Phytotherapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Transforming Growth Factor beta1 ; biosynthesis ; Treatment Outcome