Astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissues of diabetic KKAy mice

Numerous cytokines participate in the occurrence and development of inflammation and renal interstitial fibrosis. Previous studies confirmed that TGF-β1 overexpressed in diabetic nephropathy. As a downstream signal protein of TGF-β1 family, SMAD has an important role in the process of α-SMA mediated renal interstitial fibrosis. This study aimed to study astragaloside effect on TGF-β1, SMAD2/3, and α-SMA expression in the kidney tissue of diabetic KKAy mice, to reveal its potential impact on renal interstitial fibrosis. 20 type II diabetic KKAy mice were randomly equally divided into model group and astragaloside group, while 10 male C57BL/6J mice were selected as the control. Astragaloside at 40 mg/(kg•d) was given when the KKAy mice fed with high-fat diet to 14 weeks old. The mice were killed at 24 weeks old and the kidney tissue samples were collected. Pathology morphological changes were observed. TGF-β1, SMAD2/3, and α-SMA expression levels were determined by immunohistochemistry. Compared with control, mice kidney in model group appeared obvious fibrosis and up-regulated blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression (P < 0.05). Mice in astragaloside group exhibited alleviated renal interstitial fibrosis compared with the model. Its blood glucose level, TGF-β1, SMAD2/3, and α-SMA expression levels were significantly lower than the model group (P < 0.05). Astragaloside can delay the renal fibrosis process in diabetic mice by influencing the TGF-β/SMADS signaling pathway and down-regulating TGF-β1, SMAD2/3, and α-SMA expression.     

Relationship between transforming growth factor-β1 and type 2 diabetic nephropathy risk in Chinese population

Background

Diabetes mellitus (DM) is divided into four different etiological categories: type 1 DM (T1DM), type 2 DM (T2DM), other specific types, and gestational DM. One severe complication of T2DM is type 2 diabetic nephropathy (T2DN). The possible association of serum transforming growth factor-β1 (TGF-β1) levels and the TGF-β1 T869C gene polymorphism with patient susceptibility to T2DN in Chinese population is unclear at present. This study was conducted to assess these relationships in Chinese population by a meta-analysis.

Methods

Association reports were searched and pulled from the Cochrane Library, the China Biological Medicine Database (CBM), and PubMed on March 1, 2018, and eligible studies were selected and used for calculations. The results were expressed as weighted mean differences (MD) for continuous data. Odds ratios (OR) were used to express the results for dichotomous data. Additionally, 95% confidence intervals (CI) were calculated.

Results

Forty-eight reports for the relationship between serum TGF-β1 levels and the risk of T2DN and 13 studies on the association of the TGF-β1 T869C gene polymorphism with susceptibility to T2DN in Chinese population were retrieved from this study. Serum TGF-β1 levels in the T2DM group were higher than those in the normal control group (MD?=?17.30, 95% CI: 12.69–21.92, P?<?0.00001). The serum TGF-β1 level in the T2DN group was significantly higher than that in the normal control group (MD?=?70.03, 95% CI: 60.81–79.26, P?< 0.00001;). The serum TGF-β1 level in the T2DN group was significantly higher than that in the T2DM group (MD?=?56.18, 95% CI: 46.96–65.39, P?< 0.00001). Serum TGF-β1 levels in T2DM patients with microalbuminuria were increased when compared with those in T2DM patients with normoalbuminuria. Furthermore, serum TGF-β1 levels in T2DM patients with macroalbuminuria were increased when compared with those in T2DM patients with microalbuminuria. The TGF-β1 T allele, TT allele and CC genotype were associated with T2DN susceptibility in Chinese population (T: OR?=?0.74, 95% CI: 0.59–0.92, P?=?0.007; TT: OR?=?0.55, 95% CI: 0.31–0.96, P?=?0.04; CC: OR?=?1.38, 95% CI: 1.14–1.67, P?=?0.001).

Conclusions

High levels of TGF-β1 are associated with susceptibility to T2DM, T2DN and the progression of proteinuria in T2DN patients in Chinese population. Further, the TGF-β1 T allele, and TT genotype were protective factors against the onset of T2DN and CC genotype was a risk factor for the susceptibility of T2DN in Chinese populations.

     

Tumor-infiltrating lymphocytes and PD-L1 in breast cancer (and,what happened to medullary carcinoma?)

Tumor-infiltrating lymphocytes (TILs) and PD-L1 have emerged as important immune biomarkers in breast cancer, particularly triple negative breast carcinomas (TNBC) and human epidermal growth factor-2 positive (HER-2⁺) breast carcinomas. These components of the tumor immune microenvironment can be harnessed or targeted with immunotherapy, which represents a significant advancement in the management of TNBC. TILs are a prognostic biomarker in breast cancer, and this recognition has led to reclassification of medullary carcinoma (which were TILs rich by definition) as a pattern of invasive ductal carcinoma (no special type) rather than a distinct histologic type. PD-L1 is a predictive biomarker in TNBC, and two different PD-L1 assays have been approved as companion diagnostics for immune checkpoint inhibition in TNBC. This review will cover the roles of TILs and PD-L1 testing in breast cancer, both of which provide important clinical information to guide patient prognosis and therapy. This is a rapidly evolving and exciting field with significant implications for patient care.     

Differential susceptibility to lethal endotoxaemia in mice deficient in IL-1α, IL-1β or IL-1 receptor type I

Joosten LAB, van de Veerdonk F, Vonk AG, Boerman OC, Keuter M, Fantuzzi G, Verschueren I, van der Poll T, Dinarello CA, Kullberg BJ, Van der Meer JWM, Netea MG. Differential susceptibility to lethal endotoxaemia in mice deficient in IL‐1α, IL‐1β or IL‐1 receptor type I. APMIS 2010; 118: 1000–7. The role of intereukin‐1 (IL‐1) in mortality caused by endotoxaemia remains controversial. While IL‐1 receptor antagonist (IL‐1Ra) protects mice from lethal endotoxaemia, mice deficient in IL‐1β (IL‐1β^{? /?}) display normal susceptibility to lipopolysaccharide (LPS). The aim of this study was to identify the source of these discrepancies. Mice deficient in IL‐1α, IL‐1β or IL‐1R type I were injected intraperitoneally with Escherichia coli or Salmonella typhimurium LPS. Survival of the mice was examined and compared with C57/Bl6 wild‐type mice. In addition, serum cytokine concentrations were determined after LPS challenge and in vitro cytokine production by peritoneal macrophages was analysed. Clearance of radioactive IL‐1α was examined in IL‐1α^?/? and wild‐type mice. IL‐1β^?/? mice were normally susceptible to endotoxaemia and cytokine production did not differ from that in control mice. Surprisingly, LPS mortality in IL‐1α^?/? mice was significantly greater than that in control mice, accompanied by higher interferon‐γ release. These effects were mediated by a distorted homeostasis of IL‐1RI receptors, as shown by a strongly delayed clearance of IL‐1α. In contrast to the IL‐1α^?/? and IL‐1β^?/? mice, IL‐1RI^?/? mice were completely resistant to high doses of LPS. In conclusion, IL‐1RI‐mediated signals are crucial in mediating mortality occurring as a result of lethal endotoxaemia. Investigation of IL‐1‐mediated pathways in IL‐1 knock‐out mice is complicated by a distorted homeostasis of IL‐1Rs.     

The critical role of IL-12 and the IL-12Rβ2 subunit in the generation of pathogenic autoreactive Th1 cells

Experimental Allergic Encephalomyelitis (EAE) is a demyelinating disease of the central nervous system which is an animal model for the human autoimmune disease, multiple sclerosis. EAE is mediated by CD4⁺ T cells and the T cells responsible for disease induction produce Th1 cytokines. IL-12 produced by monocytes and dendritic cells is the most critical factor which influences the development and differentiation of pathogenic autoreactive Th1 cells. Here, we review our recent studies on the critical contributions of IL-12 and the IL-12Rβ2 subunit to the generation of autoreactive effector cells which mediate EAE. In addition, we discuss the potential contribution of IL-18 to the upregulation of the IL-12/IL-12Rβ2 pathway and the contribution of the suppressor cytokines, IL-4 and IL-10, in downregulating this pathway. Collectively, our studies demonstrate that the IL-12/IL-12Rβ2 pathway is a critical intermediary in the process of Th1 differentiation which can be both positively or negatively regulated. This pathway remains an attractive immunotherapeutic target for blockade of function with inhibitory reagents or downregulation by Th2 cytokines.     

Astragaloside inhibits IL-1β-induced inflammatory response in human osteoarthritis chondrocytes and ameliorates the progression of osteoarthritis in mice

Abstract

Background: Osteoarthritis (OA) is a chronic joint-degeneration disease and accounts for the most frequent arthritis in aging people. OA is characterized by the degeneration of articular cartilage, subchondral bone sclerosis and synovitis. Inflammation as an important role in OA progression, in that anti-inflammatory agents could effectively inhibit the development of OA with minimal side effects, therefore developing a nature anti-inflammatory compound will be a promising therapy for treating OA.

Methods: We treated patient-derived chondrocytes and mouse models of OA with astragaloside, an effective component of astragalus membranaceus, and measured its effect on pro-inflammatory cytokines and OA progression in mice.

Results: In vitro, astragaloside induced a dose-dependent inhibition of IL-1β-induced the production of inflammatory factors, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), nitric oxide (NO), prostaglandin E2 (PGE2), expression of MMP 13 and ADAMTS-5, and the activation of NF-κB signaling. In vivo, astragaloside ameliorate the degeneration of cartilage in mouse model of OA.

Conclusion: Astragaloside potentially serve as a promising and effective therapeutic agent for treating OA patients.     

Biodistribution of synthetic thymosin α1 in the serum,urine and major organs of mice

Thymosin fraction 5 (TF5), a thymic preparation, has been shown to be an immune-potentiating agent consisting of biologically active polypeptide components with hormone-like activities. Thymosin α₁ (Tα₁) was the first biologically active polypeptide to be purified from TF5 and completely characterized. It is an acidic peptide with an isoelectric point of 4.2 and a molecular weight of 3108. Tα₁ is considered a biological response modifier which amplifies T-cell immunity. In the present study, we have studied some pharmacokinetic properties of Tα₁ by measuring its concentrations in serum, urine and ten major organs of female Swiss-Webster mice following administration of 500 μg Tα₁ intraperitoneally. Using a modified enzymatic immunoassay, our data show a significant increase of Tα₁ in serum 2min after injection and lasting for 2 h (average: 1.55 ± 0.27μg/ml). In urine, at four different time points after injection (20min, 40 min, 2h, 6 h), increased concentrations of Tα₁ were found between 24.2 and 25.4μg/ml (average: 25 ± 0.47 μg/ml). Of the 500μg Tα₁ administered to mice, 8.97% was recovered at the end of the study, of which 2% corresponded to urine, 1.25% to serum (2 ml of serum per mouse), and 5.72% to organs. Since the urine/day volume and the serum volume of any Swiss-Webster mouse is ca 2 ml, additional extrapolation of the above mentioned values could show percentages of recovery close to 40% for urine and 2.5% for serum. In most of the organs, the wet weight concentrations of Tα₁ increased significantly during the first 40 min after injection in comparison to their baseline wet weight concentrations. These organs consisted of the following: thymus (33.1 ± 3.5 μ/g vs 18 μ/g baseline); lungs (7.7±1.1 μg/g vs 1.9 μg/g baseline); spleen (15.6±0.7 μg/g vs 5.6 μg/g); kidneys (6.2±1.1μg/g vs 3.9 μg/g); ovaries (9.2±1.4 μg/g vs 0 μg/g); and peritoneal fat (4±1 μg/g vs 0 μg/g). No significant increases were observed in the liver (1.7±0.1μg/g vs 1.4μg/g) and heart (0.7±0.5μg/g vs 0 μg/g). Increased concentrations of Tα₁ were not detected in the brain and skeletal muscle tissues. These pharmacokinetic studies of Tα₁ in mice indicate that rapid renal excretion of Tα₁ represents a major source of humoral loss following IP administration. Recent preliminary studies in humans confirm that the kidney rapidly releases high levels of Tα₁ in urine in a time frame consistent with that observed in mice.     

Does the presence of Klebsiella pneumoniae carbapenemase and New Delhi metallo-β-lactamase-1 genes in pathogens lead to fatal outcome?

Introduction: Infections due to multidrug-resistant (MDR) pathogens are a medical challenge. There is considerable apprehension among clinicians regarding pathogens reported as carrying New Delhi metallo-β-lactamase-1 (NDM) and Klebsiella pneumoniae carbapenemase (KPC) genes from their patients. In the face of extremely high rates of antimicrobial resistance, it is essential to gauge the clinical significance of isolation of pathogens carrying these genes from clinical samples. This study compares the outcome of patients infected with pathogens carrying NDM/KPC genes versus those without these genes. Methods: The study was conducted over a 1-year period at a Level-1 trauma centre. Hospital-acquired infections were diagnosed on the basis of CDC’s criteria. The correlation of isolation of a multi-resistant pathogen carrying KPC or NDM genes with the clinical outcome was ascertained. Results: A total of 276 consecutive patients admitted to the Intensive Care Units/wards of the JPNA Trauma Centre were included in this study. Of the 371 isolates recovered from these patients, 116 were from patients who had a fatal outcome. The difference in prevalence of bla_NDM and bla_KPC was not significant in any genera of Gram-negative pathogens isolated from patients who survived versus those who had a fatal outcome. Conclusion: Isolation of MDR pathogens carrying NDM/KPC genes from clinical samples is not always a harbinger of a fatal outcome. Efforts should be made to prevent cross-transmission of these pathogens.     

Angiotensin type 1 (AT1) and type 2 (AT2) receptors mediate the increase in TGF-β1 in thyroid hormone-induced cardiac hypertrophy

Increased thyroid hormone (TH) levels are known to induce cardiac hypertrophy. Some studies have provided evidence for a functional link between angiotensin II (ANG II) and transforming growth factor β1 (TGF-β1) in the heart, both being able to also induce cardiac hypertrophy. However, the contribution of this growth factor activated directly by TH or indirectly by ANG II in cardiac hypertrophy development remains unknown. To analyze the possible role of TGF-β1 in cardiac hypertrophy induced by TH and also to evaluate if the TGF-β1 effect is mediated by ANG II receptors, we employed Wistar rats separated into control, hypothyroid (hypo) and hyperthyroid (T4 − 10) groups combined or not with ANG II receptor blockers (losartan or PD123319). Serum levels of T3 and T4, systolic pressure and heart rate confirmed the thyroid state of the groups. The T4 − 10 group presented a significant increase in cardiac TGF-β1 levels; however, TGF-β1 levels in the hypo group did not change in relation to the control. Inhibition of the increase in cardiac TGF-β1 levels was observed in the groups treated with T4 in association with losartan or PD123319 when compared to the T4 − 10 group. These results demonstrate for the first time the TH-modulated induction of cardiac TGF-β1 in cardiac hypertrophy, and that this effect is mediated by ANG II receptors.     

The influence of the HLA-DRB, HLA-DQB and polymorphic positions of the HLA-DRβ1 and HLA-DQβ1 molecules on risk of Iranian type 1 diabetes mellitus patients

Type 1 Diabetes mellitus (T1D) is an autoimmune and multifactorial disease. HLA-DRB1 and DQB1 loci have the strongest association with T1D. This study aimed at investigating (i) susceptibility or protection of alleles, genotypes and haplotypes of HLA-DRB1 and DQB1 loci; and (ii) highly polymorphic amino acid residues of HLA-DRβ1 and DQβ1 in 105 Iranian T1D patients and 100 controls. The results indicated that DRB1*04:01, 03:01, DQB1*03:02, 02:01 alleles, DRB1*03:01/04:01, 03:01/13:03, DQB1*02:01/03:02 genotypes, DRB1*04:01-DQB1*03:02, DRB1*03:01-DQB1*02:01, DRB1*07:01-DQB1*03:03 haplotypes had positive association with T1D. In contrast, HLA-DRB1*15:01, 13:01, DQB1*03:01, 06:01 alleles, DRB1*11:01/15:01, DQB1*03:01/06:01, 03:01/05:01 genotypes and DRB1*15:01-DQB1*06:01, DRB1*11:01-DQB1*03:01 haplotypes had negative association with T1D. Analysis of amino acid sequence of HLA-DRβ1 and DQβ1 revealed that DRβ1(Lys71+) and DQβ1(Asp57-) were significantly more frequent in patients than in controls and had a positive effect in the development of T1D. Haplotype analysis demonstrated that HLA-DRB1(Lys71+) allele provided major susceptibility for T1D, and DQβ1(Asp57-) had an additive effect. We designed an allele-specific primer to develop an easy, quick and cost-benefit method to detect the DRβ1(Lys71+) . This method can identify all 114 DRB1 alleles encoding DRβ1(Lys71+) by three PCR reactions. The PcPPV and PcNPV were also calculated to determine the impact of HLA genotype testing at amino acid positions. It showed that the DRβ1(Lys71+/+) genotype carrier had 1% absolute risk of developing T1D.     

Cyclooxygenase 2, toll-like receptor 4 and interleukin 1β mRNA expression in atherosclerotic plaques of type 2 diabetic patients

Objectives and design

Inflammation has a prominent role in the development of atherosclerosis. Type 2 diabetes could contribute to atherosclerosis development by promoting inflammation. This status might accelerate changes in intrinsic vascular wall cells and favor plaque formation. Cyclooxygenase 2 (COX-2) is highly expressed in atherosclerotic plaques. COX-2 gene expression is promoted through activation of toll-like receptor 4 (TLR4) and pro-inflammatory cytokine interleukin 1β (IL1-β). Aim of this study is to investigate whether expression profiles of pro-inflammatory genes such as COX-2, TLR4 and IL1-β in atherosclerotic plaques are altered in type 2 diabetes (T2D).

Methods

Total RNA was isolated from plaques of atherosclerotic patients and expression of COX-2, TLR4, IL1-β analyzed using real-time PCR. Histological analysis was performed on sections of the plaque to establish the degree of instability.

Results

Statistically significant differences in mRNA expression of COX-2 and IL1-β were found in plaques of T2D compared with non-T2D patients. A multi-variable linear regression model suggests that COX-2 mRNA expression is affected by T2D pathology and IL1-β mRNA expression in atherosclerotic plaques.

Conclusions

Our results support the hypothesis that T2D pathology contributes in vivo to increase the inflammatory process associated with the atherosclerotic plaque formation, as shown by an increment of COX-2 and IL1-β mRNA expression.     

Effects and relationship of ERK1 and ERK2 in interleukin-1β-induced alterations in MMP3, MMP13, type II collagen and aggrecan expression in human chondrocytes

Interleukin (IL)-1β plays an important role in the pathogenesis of osteoarthritis and catabolic processes in articular cartilage. Growing evidence suggests that ERK1/2 activation is involved in IL-1β-mediated matrix metalloproteinase (MMP) 3, MMP13, type?II collagen and aggrecan expression in chondrocytes. To investigate the respective effects and the relationship of ERK1 and ERK2, knockdown of ERK1 and/or ERK2 was performed in human chondrocytes using specific small interfering RNAs (siRNAs), and the cells were treated with IL-1β (10?ng/ml) for 24?h. Uninfected chondrocytes treated with IL-1β (10?ng/ml) were used as a positive control. Other cells cultured without IL-1β or siRNA treatment were used as a negative control. The mRNA levels of MMP3, MMP13, type?II collagen and aggrecan were evaluated by quantitative real-time PCR. The protein levels of MMP3 and MMP13 in the culture medium were examined by ELISA. The protein levels of type?II collagen, aggrecan, ERK1/2 and phospho-ERK1/2 were evaluated by western blotting. The results indicate that IL-1β enhances MMP3 and MMP13 expression and inhibits type?II collagen and aggrecan expression. Activation of the MAPK/ERK pathway was observed. Knockdown of ERK1 or ERK2 significantly reversed these effects to similar degree. Combined knockdown of ERK1 and ERK2 displayed synergistic effects. ERK1 and phospho-ERK1 or ERK2 and phospho-ERK2 were inhibited by knockdown of ERK1 or ERK2, respectively. No compensatory effect by up-regulation of the opposite isoform was observed. The combined knockdown suppressed ERK1/2 and phospho-ERK1/2. The data suggest that although inhibition of both ERK1 and ERK2 is more effective, inhibition of either ERK isoform may be sufficient and could be used for novel therapies or as drug targets for pharmacological intervention in cartilage breakdown in osteoarthritis.     

TGF-β1-treated ADSCs-CM promotes expression of type I collagen and MMP-1, migration of human skin fibroblasts, and wound healing in vitro and in vivo

Conditioned medium from adipose-derived stem cells (ADSCs) stimulates both collagen synthesis and migration of dermal fibroblasts. However, it is still unknown whether conditioned media from tumor growth factor (TGF)-β1-treated ADSCs (TGF-β1-treated ADSCs-CM) induces increased expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and migration as well as cell cycle regulatory proteins in fibroblasts, compared to non-treated ADSCs-CM. Our data showed that TGF-β1-treated ADSCs-CM promoted effectively the proliferation and migration of human skin fibroblasts, compared to non-treated ADSCs-CM. In addition the expression of MMP-1 were markedly increased by treatment of TGF-β1-treated ADSCs-CM in fibroblasts, compared to non-treated ADSCs-CM. Expression of type I collagen protein were slightly increased by treatment of TGF-β1-treated ADSCs-CM in fibroblasts. The expression of cell cycle regulators of G1/S phase transition were not markedly altered by treatment of TGF-β1-treated ADSCs-CM. Finally, artificial wounds were made using a 4-mm punch biopsy in hairless mice and TGF-β1-treated ADSCs-CM were injected into the wound area. The injection of TGF-β1-treated ADSCs-CM promoted the wound healing process in hairless mice. Taken together, our data indicated that TGF-β1-treated ADSCs-CM induced up-regulation of type I collagen and MMP-1, promoted the migration of skin fibroblasts, and thereby promoted the wound healing process in vivo. Our data indicate that TGF-β1-treated ADSCs-CM will be a component for a wound healing accelerating agent.     

Increased circulating levels of SDF-1 (CXCL12) in type 2 diabetic patients are correlated to disease state but are unrelated to polymorphism of the SDF-1β gene in the Iranian population

Several environmental and genetic factors are believed to influence the onset of diabetes and its complications. It has also been established that cytokines play a key role in the pathogenesis of type 2 diabetes. Previous studies have revealed that the polymorphism at the stromal-derived factor 1β (SDF-1β) 3′A regulates the expression of SDF-1 (CXCL12). This study was aimed to explore this polymorphism in parallel with SDF-1 serum levels in type 2 diabetic patients. In this assessment, peripheral blood samples were collected from 200 type 2 diabetic patients and 200 healthy controls. DNA was extracted, and a PCR-RFLP screening was applied to examine the SDF-1β 3′A polymorphism. We also applied the ELISA technique to measure serum levels of SDF-1. Our results showed that there were no significant correlations between SDF-1β 3′Α polymorphism in type 2 diabetic patients when compared to controls. However, our results showed that the serum levels of SDF-1 were significantly increased in the patients when compared to controls. Based on the results of this study, we concluded that SDF-1β 3′Α polymorphism does not play a role in the pathogenesis of type 2 diabetes but that elevated serum levels of SDF-1 may be important for the etiology of type 2 diabetes but are unrelated to the SDF-1β 3′Α polymorphism.