Exposure to chronic psychosocial stress is a risk factor for various pulmonary diseases. In view of the essential role of dipeptidyl peptidase 4 (DPP4) in animal and human lung pathobiology, we investigated the role of DPP4 in stress-related lung injury in mice. Eight-week-old male mice were randomly divided into a non-stress group and a 2-week immobilization stress group. Non-stress control mice were left undisturbed. The mice subjected to immobilized stress were randomly assigned to the vehicle or the DPP4 inhibitor anagliptin for 2 weeks. Chronic stress reduced subcutaneous and inguinal adipose volumes and increased blood DPP4 levels. The stressed mice showed increased levels in the lungs of genes and/or proteins related to oxidative stress (p67phox, p47phox, p22phox and gp91phox), inflammation (monocyte chemoattractant protein-1, vascular cell adhesion molecule-1, and intracellular adhesion molecule-1), apoptosis (caspase-3, -8, -9), senescence (p16INK4A, p21, and p53) and proteolysis (matrix metalloproteinase-2 to -9, cathepsin S/K, and tissue inhibitor of matrix metalloproteinase-1 and -2), and reduced levels of eNOS, Sirt1, and Bcl-2 proteins; and these effects were reversed by genetic and pharmacological inhibitions of DPP4. We then exposed human umbilical vein endothelial cells in vitro to hydrogen peroxide; anagliptin treatment was also observed to mitigate oxidative and inflammatory molecules in this setting. Anagliptin can improve lung injury in stressed mice, possibly by mitigating vascular inflammation, oxidative stress production, and proteolysis. DPP4 may become a new therapeutic target for chronic psychological stress-related lung disease in humans and animals.

The Chinese herbal granule Tangshen Formula (TSF) has been proven to decrease proteinuria and improve estimated glomerular filtration rate (eGFR) in diabetic kidney disease (DKD) patients. However, the underlying mechanism of TSF on treatment of diabetic nephropathy (DN) remains unclear. The present study aimed to identify the therapeutic target of TSF in diabetic renal injuries through microarray-based gene expression profiling and establish its underlying mechanism. TSF treatment significantly attenuated diabetic renal injuries by inhibiting urinary excretion of albumin and renal histological injuries in diabetic (db/db) mice. We found that PLZF might be the molecular target of TSF in DN. In vivo, the db/db mice showed a significant increase in renal protein expression of PLZF and collagen III, and decrease in renal autophagy levels (downregulated LC3 II and upregulated p62/SQSTM1) compared to db/m mice. The application of TSF resulted in the downregulation of PLZF and collagen III and upregulation of autophagy level in the kidneys of db/db mice. In vitro, TSF reduced high glucose (HG)-induced cell proliferation for NRK52E cells. Further studies indicated that the exposure of NRK52E cells to high levels of glucose resulted in the downregulation of cellular autophagy and upregulation of collagen III protein, which was reversed by TSF treatment by decreasing PLZF expression. In conclusion, TSF might have induced cellular autophagy by inhibiting PLZF expression, which in turn resulted in an increase in autophagic degradation of collagen III that attenuated diabetic renal injuries.

Background: Diabetic retinopathy (DR), a kind of diabetic microvascular complication, is the leading cause of visual impairment in adults aged 30 to 65 years. Despite rapid research progress, robust predictors to assess prospectively with high precision the risk for DR in individuals with diabetes are still lacking. We investigated the relationship between pyrimidine metabolites and disease, and find out the potential biomarkers for diagnosis.Methods: The study group consisted of 116 subjects who were divided to 3 groups: control (n = 41), type 2 diabetes without retinopathy (DM, n = 37), and with retinopathy (DR, 38). Biochemical and clinical parameters, concentrations of related metabolites, including of cytosine, cytidine, uridine, thymine, thymidine and 2'-deoxyuridine were measured in plasma of all participants.Results: There was a significant increase of concentrations of cytosine (p = 0.010), cytidine (p < 0.001) and thynidine (p<0.001) with DR compared to DM. The concentration of uridine, thymine and 2'-deoxyuridine did not change.Conclusions: The concentrations of cytosine, cytidine and thynidine may be useful for monitoring the progression of DR and evaluating the treatment. And cytidine has good sensitivity and specificity for diagnosis. (C) 2011 Elsevier B.V. All rights reserved.

Metabolism of free fatty acids(FFAs) is related to several important physiological events and therefore their quantitaion in biological samples arouses extensive interest and efforts. Existing gas chromatography with flame ionization detector(GC-FID) methods for the analysis of FFAs normally require derivatization of them in order to lower boiling points. But this extra procedure tends to induce additional error and it is laborious and time-consuming. A derivatization-free method was therefore established in the present investigation to determine FFAs in human plasma by capillary(GC-FID). After extraction of FFAs from plasma, a highly polar FFAP(free fatty acid in plasma) column was employed to directly quantitate FFAs concentration, free from derivatization reaction. All sample pre-treatments were carried out at room temperature, improving recovery of short-chain FFAs. Heptadecanoic acid(C17:0) was employed as internal standard, and the proposed method was validated for recovery, precision, sensitivity, stability, and linearity. Validation data show that it is suitable for clinical study that has been applied to the evaluation of FFAs levels in plasma of diabetic nephropathy(DN) patients during the course of treatment. Forty-seven patients diagnosed with DN were admitted to the double-blind experiment. Control group(n=17) underwent solely basic treatment and the patients did not show significant change in FFAs concentration during six months of treatment. Experiment group(n=30) was supplied with traditional Chinese medicine besides basic treatment. After six months of medication, their plasma concentration of palmitic acid(C16:0), stearic acid(C18:0) and oleic acid(C18:1n-9) decreased while linolenic acid(C18:3n-3) increased significantly(P<0.05). These four compounds could be served as biomarkers in the evaluation of drug efficacy, and their quantitation in plasma may provide additional information for disease progression in DN patients.

The pathological development of diabetic kidney disease (DKD) might involve metabolic perturbations in kidney tissue. The present study was designed to detect the systematic alterations of renal cortex metabolites thereby exploring the related mechanisms of DKD development and fosinopril treatment. Based on combined gas chromatography/time-of-flight mass spectrometry (GC-TOF MS) and liquid chromatography/time-of-flight mass spectrometry (UPLC-TOF MS) data acquiring platform, we have performed a metabolomic analysis of perfused renal cortex samples from the diabetic rats induced by streptozocin and treated with or without fosinopril, a pharmacological inhibitor of angiotensin II converting enzyme (ACEI). We identified a number of abnormal metabolites in the diabetic kidney, including groups of amino acids, carbohydrates, polyols, lyso-phospholipids, glucuronides and other unidentified metabolites. Of them, an increase in intrarenal organic toxins including uremic toxins, glucuronides and glucotocixity-associated metabolites are highly correlated with diabetic kidney injury including 24h urinary protein levels and tubulointerstitial injury index. Treatment with fosinopril significantly attenuated diabetic kidney injury, and simultaneously blocked the intrarenal accumulation of these organic toxins, especially hippurate and glucuronides. These results indicate that intrarenal accumulation of organic toxins may be significant for the development of DKD and the related mechanisms deserve to be further investigated.

Diabetic nephropathy (DN) is one of the most important complications of diabetic patients and is characterized histologically by an accumulation of extracellular matrix (ECM) protein in the glomerular mesangium. Therefore, mesangial cells likely play an important role in the development of diabetic nephropathy. Here, we employed proteomic techniques to investigate the protein profile of rat mesangial cells under high-glucose culture conditions. Primary isolated rat glomerular mesangial cells were cultured under different concentrations of glucose (5.4 mmol.L-1 for normal control and 30 mmol.L-1 for high glucose) for 0, 8, 16, and 72 h, as well as for 25 days. Cellular total proteins were isolated from these cells and employed for two-dimensional gel electrophoresis (2-DE). Differentially expressed proteins were identified by matrix-assisted laser desorption - ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and some of these proteins were documented in rat models of diabetes by Western blot. Rat mesangial cells were successfully isolated in the laboratory and their proliferation rates were significantly inhibited by high glucose. Two-dimensional gel electrophoresis analyses revealed 28 differentially expressed protein spots between the normal and high-glucose groups. After MALDI-TOF-MS analysis, all 28 protein spots were successfully identified with the peptide mass fingerprint (PMF) method. Representatively, SOD1, PCBP1 and PSMA6 were validated by Western blot analysis following protein extractions from the normal and high-glucose groups. Abundance of these proteins was consistent with that found in 2-DE. Moreover, expression of SOD1, PCBP1, and PSMA6 in renal cortex was further examined in two rat models of diabetes (streptozotocin-induced and spontaneous OLETF diabetic models). Abundance of SOD1 and PCBP1 proteins did not show any significant difference between normal control and diabetic rats. However, abundance of the PSMA6 protein was significantly reduced in the renal cortex of both STZ-induced and spontaneous OLETF diabetic rats. Proteomic analysis identified 28 differentially expressed proteins in primary isolated rat mesangial cells between normal and high glucose treatments. Expression of one identified protein was found to be consistent with expression in the renal cortex of two rat diabetic models. Therefore, identification of protein expression patterns in mesangial cells can be employed to develop a therapeutic target for treatment of diabetic nephropathy.

<正>柴黄益肾颗粒为本院在研的中药新药复方制剂。由黄芪、柴胡、穿山龙、当归等药味组成。具有益气活血,清热利湿功能,用于治疗各种原因引起的慢性肾炎,伴蛋白尿或浮肿。

A sensitive and specific method was developed for simultaneous determination of 21 compounds related to the diabetic nephropathy (DN) in a single analysis using high-performance liquid chromatography coupled to ultraviolet and tandem mass spectrometry (HPLC–UV/MS/MS) in human plasma. With retention times and MS/MS for peak identification, both UV and MS detectors were used for quantification. Calibration curves suitable for the analysis of plasma were linear (r2>0.998) with limits of detection (LOD) from 10 to 1000ng/mL. Intraday relative standard deviation (R.S.D.) and interday R.S.D. were both lower than 15%. With the case and control study, we found five potential biomarkers of DN, including adenosine, inosine, uric acid, xanthine and creatinine.

The constituents in effective groups of Chinese multiherb remedy possess the similar chemical and structural characters, and the same neutral loss under collisional induced dissociation(CID). Based on this, the fingerprints of the multiherb remedy and the effective extracts Were established by using HPLC-ESI-MSn in this paper, the information of retention time, molecular weights and the fragment information was compared to confirm the attribution of effective constituents to the peaks in the fingerprint of Chinese multiherb remedy, then, the difference analysis of mass spectra was employed to deal with the fragment data for rapidly identifying the effective constituents according to the neutral loss. Taking Chinese. multiherb remedy Chailin-tang for example, its effective fractions flavonoids in Radix Scutellariae and Saikosaponins in Radix bupleuriand were studied.