Hyp Levels Research Articles

Transcriptomics and proteomics analysis of system-level mechanisms in the liver of apigenin-treated fibrotic rats

AimsLiver fibrosis is a crucial pathological feature which could result in cirrhosis and hepatocarcinoma. But until now, there is no favourable treatment for it. Apigenin (APG) is a flavonoid, which exhibits efficient anti-liver fibrosis activity, but its underlying mechanisms were rarely studied. So this work aims to estimate the potential therapeutic action of APG on liver fibrosis rats and to gain insight into its system-level mechanisms. Main methodsHepatic fibrosis was induced by CCl4 in Wistar rats, and APG was given in the light of the regimen. Biochemical indexes, histopathological change and immunohistochemistry of liver were evaluated. The optimal effect group of APG was selected for further transcriptomic and proteomic analysis. Key findingsAPG ameliorated liver fibrosis via reducing the levels of AST, ALT, ALP, LDH, Hyp, TP, TB, DB, HA, LN, PCIII and IV-C, mitigating fibrosis and inflammation of liver in H&E and Masson staining. Mechanistically, APG elevated the activity of ALB, SOD and GSH-PX with reducing the level of MDA. The results of microarray and TMT revealed that 4919 genes and 4876 proteins were differentially expressed in the APG and model groups. Besides, transcriptomics and proteomics analyses unfolded 120 overlapped proteins, enriched in 111 GO terms containing apoptotic process, angiogenesis, cell migration and proliferation, etc. Meanwhile, KEGG pathway analysis showed that 26 pathways containing HIF-1/MAPK/eNOS/VEGF/PI3K/Akt signaling pathway, regulation of actin cytoskeleton and focal adhesion mostly. SignificanceAPG can ameliorate CCl4-induced liver fibrosis via VEGF-mediated FAK phosphorylation through the MAPKs, PI3K/Akt, HIF-1, ROS, and eNOS pathways, which may hopefully become the anti-liver fibrosis activity of natural product.

Amelioration of paraquat-induced pulmonary fibrosis in mice by regulating miR-140-5p expression with the fibrogenic inhibitor Xuebijing.

Intravenous Xuebijing (XBJ) therapy suppresses paraquat (PQ)-induced pulmonary fibrosis. However, the mechanism underlying this suppression remains unknown. This work aimed to analyze the miR-140-5p-induced effects of XBJ injection on PQ-induced pulmonary fibrosis in mice. The mice were arbitrarily assigned to four groups. The model group was administered with PQ only. The PQ treatment group was administered with PQ and XBJ. The control group was administered with saline only. The control treatment group was administered with XBJ only. The miR-140-5p and miR-140-5p knockout animal models were overexpressed. The gene expression levels of miR-140-5p, transglutaminase-2 (TG2), β-catenin, Wnt-1, connective tissue growth factor (CTGF), mothers against decapentaplegic homolog (Smad), and transforming growth factor-β1 (TGF-β1) in the lungs were assayed with quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blot analysis. The levels of TGF-β1, CTGF, and matrix metalloproteinase-9 (MMP-9) in the bronchoalveolar lavage fluid were assessed by enzyme-linked immunosorbent assay (ELISA). Hydroxyproline (Hyp) levels and pulmonary fibrosis were also scored. After 14 days of PQ induction of pulmonary fibrosis, AdCMV-miR-140-5p, and XBJ upregulated miR-140-5p expression; blocked the expressions of TG2, Wnt-1, and β-catenin; and decreased p-Smad2, p-Smad3, CTGF, MMP-9, and TGF-β1 expressions. In addition, Hyp and pulmonary fibrosis scores in XBJ-treated mice decreased. Histological results confirmed that PQ-induced pulmonary fibrosis in XBJ-treated lungs was attenuated. TG2 expression and the Wnt-1/β-catenin signaling pathway were suppressed by the elevated levels of miR-140-5p expression. This inhibition was pivotal in the protective effect of XBJ against PQ-induced pulmonary fibrosis. Thus, XBJ efficiently alleviated PQ-induced pulmonary fibrosis in mice.

Moderate intensity exercise in hypoxia increases IGF-1 bioavailability and serum irisin in individuals with type 1 diabetes.

Aim:This study aimed to determine the effect of moderate intensity continuous exercise (Ex) and hypoxia (Hyp) on serum brain-derived neurotrophic factor (BDNF), insulin-like growth factor-1 (IGF-1) and its binding protein-3 (IGFBP-3), irisin and cytokines levels in patients with type 1 diabetes (T1D).Methods:A total of 14 individuals with T1D (age: 28.7 ± 7.3 years) and 14 healthy adults (age: 27.1 ± 3.9 years) performed 40-min continuous Ex at moderate intensity (50% lactate threshold) on a cycle ergometer in normoxia (Nor) and Hyp (FiO2 = 15.1%) Biochemical factors, glucose concentrations and physiological variables were measured at rest, immediately and up to 24 h after both Ex protocols.Results:Patients with T1D had significantly lower pre-Ex serum concentrations of BDNF (p < 0.05, p < 0.01), and total IGF-1 (p < 0.001, p < 0.05) and significantly higher irisin levels (p < 0.05, p < 0.01) in Nor and Hyp, compared with healthy subjects. Ex significantly increased in T1D group serum BDNF (in Nor only p < 0.05) and total IGF-1 levels in Nor and Hyp (p < 0.001 and p < 0.01, respectively). Immediately after Ex in Hyp, freeIGF-1 (p < 0.05) and irisin levels (p < 0.001) were significantly higher compared with the levels induced by Ex alone. Free IGF-1 and irisin serum levels remained elevated in 24 h post-Ex in Hyp. In T1D, significant blood glucose (BG) decrease was observed immediately after Ex in Hyp (p < 0.001) and in 24 h recovery (p < 0.001) compared with pre-Ex level.Conclusion:The study results suggest that moderate intensity continuous Ex has beneficial effect on BDNF and IGF-1 levels. Ex in hypoxic conditions may be more effective in increasing availability of IGF-1. The alterations in the post-Ex irisin levels and IGF-1 system may be contributing to more effective glycaemia control in patients with T1D.

Effect of hydroxyproline supplementation on growth performance, body composition, amino acid profiles, blood‐biochemistry and collagen synthesis of juvenile chu's croaker ( Nibea coibor )

Hydroxyproline (Hyp) may play an important role in collagen metabolism, as it is almost exclusively found in collagen. To examine this possibility, a 56-day feeding trial was conducted to evaluate the effect of graded supplementaries of Hyp (0, 2.5, 5, 7.5, 10 and 12.5 g/kg dry diet weight) on growth performance, feed utilization, biochemical composition and collagen synthesis of chu's croaker, Nibea coibor (13.6 ± 0.28 g). The growth performance and feed utilization were improved with increased levels of dietary Hyp, reaching a maximum at 5 g/kg (diet H2) followed by a slight decrease. Polynomial regression analysis showed that the maximum specific growth rate (SGR) was attained at 7.177 g/kg dietary Hyp. Increasing levels of Hyp did not affect body composition of juvenile Nibea coibor. However, dietary Hyp supplementation significantly affected the levels of Leu, Ile, Met, Lys, Ser, Glu and Pro in muscle and the levels of Met, Lys, Glu, Pro and Gly in swim bladder. The serum calcium (SC) level decreased significantly with increasing levels of dietary Hyp. However, the triglycerides (TG) and total cholesterol (T-CH) levels in serum and liver were relatively stable. Importantly, the acid-soluble collagen (ASC) and total collagen (TC) levels in swim bladder increased significantly initially and then decreased after reaching a maximum. Polynomial regression analysis revealed that the maximum TC level in swim bladder was attained at 9.736 g/kg of dietary Hyp. Collectively, these results suggest that the appropriate provision of hydroxyproline is essential for maximal collagen synthesis and growth.

Effect of Ficus carica L. leaves extract loaded gold nanoparticles against cisplatin-induced acute kidney injury

BackgroundCisplatin (CisPt) is one of the most widely used and highly effective drugs for the treatment of various solid tumors, unfortunately acute kidney injury (AKI) is considered one of its side effects through several mechanisms including production of reactive oxygen species (ROS), pro-inflammatory and pro-fibrotic cytokines. Due to the poor effect of AKI therapy, the use of nanoparticles loaded with natural extracts for delivering to the kidney molecules are desirable. AimThis study aims to investigate the effectiveness of different concentrations of gold nanoparticles (Au-NPs) as a carrier for Ficus carica L. (Fig) leaves extract against CisPt induced AKI. MethodsSeventy male albino rats were used and divided into seven groups. After the experimental period, blood was withdrawn, serum was separated for determination of urea, creatinine, homocystein (Hcy) and folic acid while reduced glutathione (GSH), nitric oxide (NO), malondialdehyde (MDA), total antioxidant capacity (TAC) and hydroxyproline content (Hyp) were evaluated in kidney tissue homogenate. ResultsCisPt induced AKI in rats and results in a significant increase in the levels of serum urea, creatinine, Hcy and kidney Hyp, lipid peroxidation along with a significant reduction of kidney GSH, NO and TAC compared to the control rats. Treatment with Au-NPs and Fig extract particularly in a ratio of (3:2) respectively was shown to improve renal functions with efficient capacity in scavenging ROS and reduced AKI severity. ConclusionAu-NPs enhanced the anti-oxidative properties of the Fig extract in targeting kidney damaged tissue and reduced oxidative toxicity induced by CisPt.

Dexamethasone combined with berberine is an effective therapy for bleomycin-induced pulmonary fibrosis in rats.

Pulmonary fibrosis (PF) is a heterogeneous pathological process in lung tissues with a considerable mortality rate. Currently, combination therapy represents an effective approach to treat PF. Dexamethasone (Dxs) and berberine (BBR) are widely applied to inhibit the progression of PF. Dxs plus penehyclidine hydrochloride or alfacalcidol have been reported more effective in therapy compared with any single drug treatment. However, whether Dxs plus BBR induces an increased antifibrotic effect remains unknown. The current study aimed to evaluate the therapeutic effect of BBR plus Dxs in bleomycin (BLM)-induced PF. A PF model in rats was established and rats were divided into control, BLM, BBR, Dxs and BBR plus Dxs groups (n=9/group). On days 3, 7 and 14, blood samples were collected from the eyes of the rats (n=6/group). CXC chemokine ligand 14 (CXCL14), collagen I, collagen III, matrix metalloproteinase (MMP)2 and MMP9 serum levels were measured by ELISA. On day 14, all rats were sacrificed. Hematoxylin and eosin analysis, Masson staining and hydroxyproline (Hyp) assessment were performed to observe histopathological changes and collagen deposition. mRNA and protein levels of CXCL14, CXC chemokine receptor 4 (CXCR4), collagen I/III, α-smooth muscle actin (α-SMA), MMP2/9 and phosphorylated-Smad 2/3 in lung tissue were further evaluated. Similar effects in preventing lung damage were observed histopathologically for Dxs and BBR compared with the BLM group. These treatments further reduced levels of Hyp, CXCL14, CXCR4, collagen I/III, MMP2/9, α-SMA and p-Smad 2/3. The combination of Dxs and BBR exhibited increased effectiveness compared with the single treatments. Results further suggested that antifibrotic mechanisms were involved in inhibiting CXCL14 and MMP2/MMP9 expression, and preventing the activation of Smad2/3 and hedgehog signaling pathways. The combined use of Dxs and BBR may represent a potential therapeutic approach for PF.

Gas Chromatography-Mass Spectrometry (GC-MS) Analysis of the Volatile Oil of Cichorium Glandulosum Boiss et Huet and its Effects on Carbon Tetrachloride-Induced Liver Fibrosis in Rats.

BackgroundThis study aimed to use gas chromatography-mass spectrometry (GC-MS) to identify the chemical constituents of volatile oil extracted by steam distillation from Cichorium glandulosum Boiss et Huet (CG), a traditional Uyghur medicine, and to investigate its effects on carbon tetrachloride (CCl4)-induced hepatic fibrosis in rats.Material/MethodsSprague-Dawley rats (n=60) included six groups: the control group (n=10), untreated model group (n=10), the volatile oil of CG high-dose group (0.15 ml/kg) (n=10), the volatile oil of CG medium-dose group (0.10 ml/kg) (N=10), the volatile oil of CG low-dose group (0.05 ml/kg) (n=10), and the silybin-treated group (0.20 ml/kg) (n=10). Rats given the essential oil extract of CG by intragastric administration, and then subcutaneously injected with a solution of CCl4 in olive oil to create the rat model of hepatic fibrosis. Serum samples were analyzed for markers of liver function, including aspartate transaminase (AST), alanine transaminase (ALT), malondialdehyde (MDA), hydroxyproline (Hyp), γ-glutamyl transpeptidase (γ-GT), lactate dehydrogenase (LDH), alkaline phosphatase (ALP), and albumin (Alb). Histology and immunohistochemistry were performed on rat liver tissue.ResultsThirty-eight compounds were identified from the volatile oil of CG (total, 98.058%), with terpenoids, including citronellol, being the most abundant. In the animal model of liver fibrosis, all doses of volatile oil of CG significantly reduced the serum levels of AST, ALT, MDA, Hyp, γ-GT, LDH, ALP, and Alb.ConclusionsGC-MS identified the components of the volatile oil of CG, which included citronellol. Treatment with volatile oil of CG reduced liver fibrosis in a rat model.

Comparative effects of horseradish (Moringa oleifera) leaves and seeds on blood pressure and crucial enzymes relevant to hypertension in rat

Horseradish (Moringa oleifera) is consumed as a vegetable and used for medicinal purposes. The effects of horseradish (Moringa oleifera) leaves and seeds on blood pressure measurement, enzymatic and non-enzymatic antioxidant status and critical enzymes [angiotensin-1 converting enzyme (ACE), acetylcholinesterase (AChE), arginase, phosphodiesterase-5 (PDE-5)] relevant to hypertension in vivo were determined. Hypertensive rats (HYP) were placed on dietary regimen containing 2% and 4% horseradish leaves and seeds. The result revealed that there were significant (p < 0.05) significant (p < 0.05) reductions in the systolic and diastolic blood pressure levels of HYP fed horseradish leaves and seeds supplemented diets when compared to the elevated levels of HYP. Furthermore, there were significant (p < 0.05) reductions in the activities of arginase, AChE, PDE-5 and ACE of HYP fed horseradish seeds and leaves supplemented diets when compared to HYP. In addition, HYP fed horseradish seeds and leaves supplemented diet had significantly (p < 0.05) higher antioxidant effect when compared to HYP. Thus, the observed salutary effects of the seeds and leaves could be part of the possible biochemical mechanisms behind their folkloric use as antihypertensive plants. However, horseradish leaves showed higher antihypertensive effects than the seeds.

Mechanism of combined use of vitamin D and puerarin in anti-hepatic fibrosis by regulating the Wnt/β-catenin signalling pathway.

AIMTo reveal the protective mechanism of the combined use of vitamin D and puerarin in the progression of hepatic fibrosis induced by carbon tetrachloride (CCl4).METHODSEight-week-old male Wistar rats were randomly divided into a normal control group (C group), a CCl4 group (CCl4 group), a vitamin D group (V group), a puerarin group (P group), and a combined group of vitamin D and puerarin (V + P group), each of which contained ten rats. In this way, we built a rat model of CCl4-induced hepatic fibrosis with intervention by vitamin D, puerarin, or a combination of the two. After eight weeks, the mice were sacrificed to collect serum and liver specimens. Blood was collected to detect the hyaluronic acid (HA). We also measured hydroxyproline (Hyp) and prepared paraffin sections of liver. After Sirius red staining, the liver specimens were observed under a microscope. RT-PCR and western blot analysis were adopted to detect the mRNA and the protein levels of Collagen I, Collagen III, Wnt1, and β-catenin in the liver tissues, respectively.RESULTSHepatic fibrosis was observed in the CCl4 group. In comparison, hepatic fibrosis was attenuated in the V, P, and V + P groups: the HA level in blood and the Hyp level in liver were reduced, and the mRNA levels of Collagen I, Collagen III, Wnt, and β-catenin in liver were also decreased, as well as the protein levels of Wnt1 and β-catenin. Among these groups, the V + P group demonstrated the greatest amelioration of hepatic fibrosis.CONCLUSIONThe combined application of vitamin D and puerarin is capable of alleviating CCl4-induced hepatic fibrosis of rats. As to the mechanism, it is probably because the combined use is able to silence the Wnt1/β-catenin pathway, suppress the activation of hepatic stellate cells, and reduce the secretion of collagen fibers, therefore improving the anti-hepatic fibrosis effect.

Effects of hydrogen sulfide on renal fibrosis in diabetic rats and its mechanism

To investigate the effects of hydrogen sulfide (H2S) on renal fibrosis in diabetic rats and explore its mechanism. Male Sprague-Dawley rats were randomly divided into normal control (NC) group, a diabetic control (DC) group, diabetes mellitus (DM)+sodium hydrosulfide (NaHS) group and DM+DL-propargylglycine (PAG) group, with 8 rats in each group.Type 1 diabetes was induced in the respective groups by a single intraperitoneal (i.p.) injection of streptozotocin.From the fifth week, rats in the DM+NaHS and DM+PAG groups were injected (i.p.) with 56 μmol/kg NaHS and 40 mg/kg PAG once a day, respectively.After treatment for 4 weeks, the levels of fasting blood glucose (FBG), blood urea nitrogen (BUN) and serum creatinine (SCr) were detected.The deposition of renal collagen fibers was observed by Masson staining, and collagen volume fraction (CVF) was calculated.The ultrastructural change of renal tissue was observed by transmission electron microscopy.The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and hydroxyproline (Hyp) in renal tissues were detected using the kits.The expression levels of TGF-β1, Smad3, phosphorylated (p)-Smad3 and collagen-IV (col-IV) in renal tissues were detected using Western blot. Compared with the NC group, the levels of FBG, BUN, SCr, CVF, IL-1β, IL-6, TNF-α and Hyp were increased; the deposition of renal collagen fibers and the ultrastructural damage were aggravated; the levels of TGF-β1, Smad3, p-Smad3, p-Smad3/Smad3 and col-IV were increased in the DC group.Compared with the DC group, excluding FBG, the aforementioned indices were improved in the DM+NaHS group; the aforementioned indices were further aggravated in the DM+PAG group. H2S attenuated renal fibrosis in diabetic rats, and the mechanism might be associated with the reduction of the release of proinflammatory cytokines, downregulation of the TGF-β1/Smad3 pathway, and inhibition of excessive accumulation of col-IV.

Melatonin Ameliorates Liver Fibrosis Induced by Carbon Tetrachloride in Rats via Inhibiting TGF-β1/Smad Signaling Pathway.

Melatonin has been reported to inhibit hepatic fibrosis and the mechanism may be correlated to its anti-oxidant effect. Nevertheless, the mechanism is not completely identified. This study was conducted to investigate the effects of melatonin on TGF-β1/Smad signaling pathway in liver fibrosis in rats. The liver fibrosis model was made by the subcutaneous injection of CCl4. The liver pathology changes were detected using hematoxylin and eosin (H&E) staining and Van Gieson (VG) staining. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities were measured with an autoanalyzer. Glutathione peroxidase (GPx) activities and levels of malondialdehyde (MDA) and hydroxyproline (Hyp) in liver were evaluated by spectrophotometry. Expression levels of TGF-β1, Smad2/3, phosphorylated Smad2/3 (p-Smad2/3) and Smad7 in liver were detected by immunohistochemistry and Western blot analysis. Results showed that melatonin suppressed CCl4-induced liver fibrosis, along with an improvement in histological changes, significant decreases in pathologic grading sores and obvious decreases in Hyp levels in liver. Melatonin improved liver function indicated by decreased serum ALT and AST activities. In addition, melatonin exerted its anti-oxidant effects, as supported by decreased MDA levels and increased GPx activities in liver. Furthermore, melatonin inhibited TGF-β1/Smad pathway, as evidenced by decreased TGF-β1, Smad2/3 and p-Smad2/3 expression and increased Smad7 expression in liver. In conclusion, melatonin may suppress CCl4-induced hepatic fibrosis in rats via inhibiting TGF-β1/Smad pathway. It is possible for melatonin to be a potential reagent to treat and cure liver fibrosis.

Galectin-1 Restores Immune Tolerance to Liver Transplantation Through Activation of Hepatic Stellate Cells

Background/Aims: Immune tolerance is considered the only way to manage liver transplantation (LT). The current study hypothesized that galectin-1 via the activation of hepatic stellate cells (HSCs) is capable of inducing immune tolerance in LT. Methods: Lentiviral-mediated gene knockdown and overexpression of galectin-1 were conducted in HSC-T6 cells. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to determine galectin-1 expression. LT was performed in 20 C57BL/J6 mice and 20 C3H mice. T-cells were assigned into control, Galectin-1 shRNA, Galectin-1 OE, Galectin-1 OE SB431542, Galectin-1 OE Sulforaphane, Galectin-1 OE Y27632, and Galectin-1 OE UO126 groups. CFSE, flow cytometry, and ELISA were respectively employed to detect T-cell proliferation, CD4+/ CD8+ ratio and IL-2, IL-10 and TGF-β levels. After establishing mouse models of immune tolerance and acute rejection, immunohistochemistry, TUNEL, and immunofluorescence assay were performed to determine CD3+ expression, apoptosis, α-SMA, and desmin. Mouse models of CCl4-induced liver fibrosis were established, followed by assigning the control1 and CCl4 groups. ELISA was used to determine ALT, AST, TBIL and Hyp levels. A total of 3 C57BL/J6 mice (donor) and 6 C3H mice (recipient) were grouped into the control2 and UO126 groups, followed by ELISA detection for IL-2, IL-10 and TGF-β. Results: In T-cells, galectin-1 shRNA increased cell proliferation and IL-2 levels with reduced IL-10 and TGF-β levels, while the Galectin-1 OE and Galectin-1 OE UO126 groups revealed the opposite results. Galectin-1 overexpression elevated the ratio of the CD4+ to CD8+ T-cells. The acute rejection group exhibited enhanced desmin expression and reduced α-SMA expression. Compared with the immune tolerance group, the acute rejection group displayed higher galectin-1 expression, a positive expression rate of CD3+ T-cells, and an increased apoptosis rate. Compared with the control1 group, the CCl4 group exhibited higher galectin-1 expression, ALT, AST, TBIL, and Hyp levels, α-SMA expression and CD4+/CD8+ T-cell ratio, in addition to decreased expression of desmin. Compared with the control2 group, UO126 increased galectin-1 expressions, IL-10 and TGF-β levels and reduced IL-2 levels with inactivated HSCs. Conclusions: The findings of the current study indicated that the overexpression of galectin-1 promoted the activation of HSCs, which reduced the inflammatory response by exerting immunosuppressive effects and accordingly contributed to immune tolerance in LT.

Antioxidant, antiapoptotic and amino acid balance regulating activities of 1,7-dihydroxy-3,4,8-trimethoxyxanthone against dimethylnitrosamine-induced liver fibrosis.

Liver fibrosis represents the consequences of a sustained wound healing response to chronic liver injury which could be caused by viral, autoimmune, drugs, and so on. Unfortunately, there was no effective therapy available for liver fibrosis in clinic. In this study, we identified the anti-fibrotic effects of 1,7-dihydroxy-3,4,8-trimethoxyxanthone (ZYC-1) on the dimethylnitrosamine (DMN)-induced rat model. ZYC-1 was isolated from Swertia punicea Hemsl and was administrated to DMN-induced rat model. ZYC decreased the hyaluronic acid (HA), type IV collagen (CIV) and hydroxyproline (Hyp) levels and inhibited the expression of α smooth muscle actin (α-SMA) and transforming growth factor beta 1 (TGF-1β). The anti-fibrotic effect of ZYC-1 was also confirmed by Sirius Red staining. Finally, we identified 42 differentially expressed proteins by using proteomics analysis after ZYC-1 treatment, of which 17 were up-regulated and 25 were down-regulated. These Most of the 42 proteins are involved in the oxidative stress pathway, the mitochondrial-mediated apoptotic pathway and the amino acid metabolism pathway. Our study presented the first elucidated mechanisms of xanthone on liver fibrosis in vivo. This study pointed out that ZYC-1 may be used as a lead compound for hepatofibrosis treatment.

Bioanalytical Method for the Determination of Hydroxyproline in Mouse Kidney by High-Performance Liquid Chromatography with Tandem Mass Spectrometric Detection.

An analytical method was developed and validated for the measurement of hydroxyproline (Hyp) levels in mouse kidney by high-performance liquid chromatography with tandem mass spectrometric detection (LC/MS/MS) using an analytical column specially designed for the LC/MS/MS analysis for intact amino acids. Tissue hydrolyzed with hydrochloric acid could be directly injected into the LC/MS/MS, as well as separated and detected using the deuterium labelled Hyp as an internal standard. The calibration curve showed good linearity from 5 to 500 nmol/mg of tissue; the precision and accuracy, including within- and between-run, were less than 6% and within 100 ± 6%, respectively.

Reduction of Abdominal Adhesions with Elecrospun Fiber Membranes in Rat Models

ABSTRACTPurpose: The aim of this study was to assess the effects three electrospun fiber membranes on postoperative adhesion formation in rat surgical models. Materials and Methods: The PEG-PHBV (polyethylene glycol and poly-β-hydroxybutyrate valerate), PLA(Polylactic acid) and CS-PEG(chitosan and polyethylene glycol) were used for submicrometric membranes fabrication by electrospinning. A total of 64 SD male rats were randomly divided into 4 groups, one control group and 3 treatment groups, each consisting of 16 rats. The rats were underwent median laparotomy and standardized abrasion of the cecum. The indicated membranes were applied intraperitoneally at the end of the surgical procedure in the treatment groups, while the controls left without treatment. Two and four weeks after surgery respectively, a relaparotomy was made for adhesion grading and histopathological examination, Hydroxyproline (Hyp) levels at the adhesion sites in each group were measured. Results: The electrospun submicrometric membranes significantly reduced not only the incidence of adhesion but also the adhesion grading scales (p < 0.05 compared to the control group) at both two weeks and four weeks after the initial operation. Furthermore, the fibrosis grades were also higher in control groups as compared with the submicrometric membrane groups. Hyp levels in the control group was significant higher than the three experiment groups at both 2 and 4 weeks time points (p < 0.05). There were no statistical differences between the different treatment groups at either time points. Conclusions: Three electrospun submicrometric membranes were soft, flexible, easy to handle, and effectively reduced adhesion formation in rat modes.

49 The ameliorating effect of Danggui Shaoyao powder on experimental diabetic nephropathy

Objectives Danggui Shaoyao powder (DSS), a Chinese herbal compound, has been used in China with established therapeutic efficacy in patients with diabetic nephropathy (DN). The purpose of this study was to investigate the possible mechanism of DSS improving DN. Methods Wistar rats with streptozotocin (STZ)-induced diabetes were used for evaluation of the effect of treatment with DSS on DN. Rats were randomly divided into three groups: control, diabetic and diabetic+DSS. Blood glucose, serum creatinine (Cr), blood urea nitrogen (BUN), superoxide dismutase (SOD) activity, malondialdehyde (MDA) and hydroxyproline (Hyp) were measured in kidney tissue. Glomerular morphology was observed by light microscopy. Immunohistochemistry and Western blot were employed to determine the proteins levels of TGF-β1 and type IV collagen. Results Compared with the control group, Cr, BUN, MDA and Hyp levels in DN rats were significantly increased but were significantly decreased by treatment with DSS. While SOD activity in renal tissue was decreased, DSS can increase SOD activity. The renal pathological changes in the DSS treatment group were ameliorated. Furthermore, the DSS decreased the expression of TGF-β1 and collagen IV protein. Conclusions These results demonstrate that DSS can ameliorate STZ-induced experimental DN. The mechanism may be related to modulating the expression of collagen IV and TGF-β1 protein. Acknowledgments This research is supported by a project grant from the National Natural Science Foundation of China (Grant No. 81603527), Science and Technology Project of Henan Province (Grant No. 162102310466), Key Scientific Research Projects of Henan Province Colleges and Universities (Grant No. 16A360010) and Henan University of Traditional Chinese Medicine Scientific and Technological Innovation Talent Support Program (Grant No. 2015XCXRC05).

Dietary hydroxyproline improves the growth and muscle quality of large yellow croaker Larimichthys crocea

The present study was conducted to estimate the effect of dietary hydroxyproline (Hyp) on growth, muscle texture and collagen content of large yellow croaker. Six isonitrogenous and isolipidic practical diets were formulated to contain graded levels of Hyp (0.17%, 0.26%, 0.33%, 0.50%, 0.69% and 0.86% of dry matter). Fish with similar size (initial body weight, 189.87±0.89g) were distributed into 18 floating net-cages (1.5m×1.5m×2.0m). Each diet was hand-fed to triplicate groups of large yellow croaker for 82days. The results showed that the specific growth rate (SGR) of fish fed diet with 0.33% of Hyp was significantly higher than those fed with the basal diet (0.17% Hyp) (P<0.05). No significant differences were found in survival rate, viscerosomatic index, hepatosomatic index, condition factor and feed intake among all treatments (P>0.05). Protein efficiency ratio and feed efficiency significantly increased with increasing levels of dietary Hyp up to 0.33% (P<0.05). Moisture and crude lipid contents in muscle showed no significant difference among all treatments (P>0.05), while crude protein was significantly improved by dietary Hyp (P<0.05). Significant decreases were observed in muscle liquid loss and water loss with increasing levels of dietary Hyp (P<0.05), while little variation was detected in lipid loss (P>0.05). A statistically significant difference was observed in muscle pH with dietary Hyp (P<0.05). Except for the cohesiveness and adhesiveness, all other analyzed texture properties including hardness, springiness, chewiness in muscle were significantly affected by dietary Hyp. Alkaline-soluble Hyp reached to the highest value in muscle when fish were fed diet with 0.69% of Hyp. The highest value of the total Hyp content in muscle was found in the treatment with 0.86% of dietary Hyp (P<0.05). No significant difference was detected in muscle alkaline-insoluble Hyp, salt-soluble protein and water-soluble protein (P>0.05) among all treatments. Pyridinium crosslink (PYD) in muscle increased with increasing dietary Hyp content up to 0.69% (P<0.05). Hardness, springiness and chewiness showed a high and positive correlation with alkaline-soluble Hyp, total Hyp and PYD, and negative correlation with liquid loss and water loss. It was concluded that dietary Hyp could benefit fish growth, promote the formation of collagen, and thereafter influence muscle quality of large yellow croaker. Using the broken-line models based on SGR and the total Hyp content in muscle, the optimal dietary Hyp content for large yellow croaker were estimated to be 0.33% and 0.61%, respectively. Statement of relevanceThis study is not a test of commercial aquaculture.

Synergistic anti-liver fibrosis actions of total astragalus saponins and glycyrrhizic acid via TGF-β1/Smads signaling pathway modulation

Ethnopharmacological relevanceHuangqi decoction (HQD) is a well-known traditional Chinese herbal formulation, It is an effective treatment for consumptive disease and chronic liver diseases. It consists of Radix Astragali (Astragalus membranceus(Fisch.) Bge. Root, Huangqi) and Radix Glycyrrhizae (Glycyrrhiza uralensis Fisch., root and rhizome, Gancao). Total astragalus saponins (AST) is a main component of Radix Astragali and glycyrrhizic acid(GA) is a main component of Radix Glycyrrhizae. Our primary results showed that the combination of AST and GA had an obvious synergistic effect in reducing liver collagen deposition and decreasing serum alanine aminotransferase (ALT) activity in dimethylnitrosamine (DMN)-induced liver fibrosis. Aim of the studyThrough in vivo and in vitro experiments, we aimed at investigating the key anti-fibrosis signal pathway TGF-β1/Smads to further explore the synergistic mechanism of AST and GA. Material and methodsTwo hepatic fibrosis animal models, bile duct ligation-induced (BDL) and DMN-induced, were utilized. Rats were treated orally with AST, GA or AST/GA, with the effects evaluated via liver histopathology, hydroxyproline (Hyp) levels, and α-SMA expression. In the hepatic stellate cell line JS-1, cells were treated with AST/GA for 24h, followed by a cell viability assessment using Cell Counting Kit-8(CCK-8) and Real-time PCR and Western blot analysis of α-SMA, ColⅠ and TGF-β1/Smads signaling pathway related components. ResultsThe AST/GA combination attenuated liver tissue inflammation, collagen deposition, Hyp levels, and α-SMA expression in both BDL-and DMN-stimulated hepatic fibrosis rats. In vitro results showed that the AST/GA combination significantly inhibited JS-1 cell viability, significantly suppressed α-SMA, ColⅠ, TGF-β1, Smad2 and Smad3 mRNA and protein expression, as well reduced p-Smad2/3. Compared with AST or GA treatment alone, the AST/GA combination significantly reduced Smad3 mRNA expression levels and TGF-β1, Smad3, and p-Smad2/3 protein levels. ConclusionsAST and GA synergistically alleviated both BDL-and DMN-induced hepatic fibrosis via TGF-β1/Smads signaling pathway inhibition in hepatic stellate cells.

HIF-1 α as a Key Factor in Bile Duct Ligation-Induced Liver Fibrosis in Rats

ABSTRACTBackground: Although several studies suggested hypoxia as an important microenvironmental factor contributing to inflammation and fibrosis in chronic liver diseases, the mechanism of this process is not fully understood. We considered hypoxia inducible factor (HIF-1α) as a key transcription factor in liver fibrosis. The aim of the study was to evaluate the mechanisms of signaling pathway during bile duct ligation (BDL)-induced liver fibrosis in rats. Methods: BDL animal model of liver fibrosis was used in the study. Male Wistar rats were divided randomly into two experimental groups: sham group (n = 15), BDL group (n = 30). Hydroxyproline (Hyp) content as a marker of collagen accumulation in liver of rats subjected to BDL was evaluated according to the method described by Gerling B et al. Expression of signaling proteins [integrin β1 receptor, HIF-1α, nuclear factor kappa B (NF-κB), and transforming growth factor (TGF-β)] was evaluated applying Western-immunoblot analysis. In all experiments, the mean values for six assays ± standard deviations (SD) were calculated. The results were submitted to the statistical analysis using the Student's “t” test, accepting p < 0.05 as significant. Results: Ligation of bile ducts was found to increase Hyp content in rat liver, accompanied by increase of HIF-1α expression during 10 weeks after BDL. The Hyp level was time dependent. There was not such a difference in control group (p < 0.001). Simultaneously expression of NF-κB, TGF-β, β1-integrin receptor was significantly elevated starting from sixth week after ligation. Activity of metalloproteinases 2 and 9 in the livers were increased 1 week after surgery and remained increased until the end of the experiment. Conclusions: The mechanism of development of liver fibrosis involves activation of Matrix metalloproteinase-2 (MMP-2) and Matrix metalloproteinase-9 (MMP-9), upregulation of HIF-1α transcriptional activity and its related factors, NF-κB and TGF-β. It suggests that they may represent targets for the treatment of the disease.

Intermittent Hypoxia Increases Mitochondrial Dynamics and Biogenesis After Eccentric Exercise-Induced Muscle Damage in Trained Rats

Mitochondria play a key role in (and are modulated by) exercise physiology, response to hypoxia and to cellular damage. Consequently, hypobaric hypoxia and exercise could play an important role in muscle damage and recovery through mitochondrial regulation. PURPOSE: To analyze the mitochondrial biogenesis and dynamics in eccentric-exercise induced muscle damage (EEIMD) and its recovery with intermittent hypobaric hypoxia (IHH), alone or combined with light exercise. METHODS: Muscle injury was induced by downhill running to forty-eight Sprague-Dawley trained rats. They were divided into three groups: (1) Ctrl (passive recovery); (2) HYP (exposed to IHH: 4-hour session per day, at 4,000 m); and (3) EHYP (IHH + light aerobic exercise). Each group was analyzed 1, 3, 7 and 14 days after the muscle damage. The following proteins were determined by Western Blotting: PGC-1α, TFAM, TOM20 (proteins related to biogenesis), Mfn1, OPA-1 and DRP-1 (proteins related to mitochondrial fusion and fission) and Sirt3 (protein related to biogenesis and oxidative stress). All results were normalised to the Ctrl group. RESULTS: 7 days after the damage induction, both HYP and EHYP groups showed significant increased levels of biogenesis markers PGC-1α (161 and 159%, respectively) and TOM20 (156 and 147%) compared with the Ctrl group, while only EHYP had a significant increase of TFAM. At t14 only EHYP rats had increased levels of PGC-1α (128%), but both HYP and EHYP groups showed increased TFAM (153 and 155%) and TOM20 (145 and 144%). Sirt3 was significantly increased in HYP t03 (200%) and in the EHYP group at t07 (127%). Regarding to the mitochondrial fusion proteins, HYP and EHYP rats had increased levels of OPA-1 at t07 (171% both) and t14 (136 and 128%), while Mfn1 was only increased at t14 in EHYP (158%). On the other hand, DRP-1 appeared elevated in HYP t07 (133%) and EHYP t14 (162%) groups. These differences had a p-value <0.05. CONCLUSIONS: after an EEIMD protocol, IHH exposure increased mitochondrial biogenesis and dynamics, as well as Sirt3 protein. When combined with light aerobic exercise, these increments were more consistent and took place in a later stage of the recovery. Taking into account the important role of the mitochondria in the muscle cell, the results suggest that these protocols can improve the muscle damage recovery.