Impact of non-alcoholic fatty liver disease and fibrosis on mortality and kidney outcomes in patients with type 2 diabetes and chronic kidney disease: A multi-cohort longitudinal study.

AIM: To evaluate the impact of non-alcoholic fatty liver disease (NAFLD) presence and fibrosis risk on adverse outcomes in patients with type 2 diabetes and chronic kidney disease. METHODS: Data were sourced from two longitudinal cohorts: 1172 patients from the National Health and Nutrition Examination Survey (NHANES) and 326 patients from the kidney biopsy cohort at the West China Hospital of Sichuan University. Cox regression estimated hazard ratios (HRs) for NAFLD and liver fibrosis concerning adverse clinical outcomes. Subsequently, a two-sample Mendelian randomization study using genome-wide association study statistics explored NAFLD's potential causal link to cardio-cerebrovascular events. RESULTS: In the NHANES cohort, NAFLD stood as an independent risk factor for various outcomes: overall mortality [HR 1.53 (95% confidence interval, CI 1.21-1.95)], mortality because of cardio-cerebrovascular diseases [HR 1.63 (95% CI 1.12-2.37)], heart disease [HR 1.58 (95% CI 1.00-2.49)], and cerebrovascular disease [HR 3.95 (95% CI 1.48-10.55)]. Notably, advanced liver fibrosis, identified by a fibrosis-4 (FIB-4) score >2.67, exhibited associations with overall mortality, cardio-cerebrovascular disease mortality and heart disease mortality. Within the kidney biopsy cohort, NAFLD correlated with future end-stage kidney disease [ESKD; HR 2.17 (95% CI 1.41-3.34)], while elevated FIB-4 or NAFLD Fibrosis Scores predicted future ESKD, following full adjustment. Liver fibrosis was positively correlated with renal interstitial fibrosis and tubular atrophy in biopsies. Further Mendelian randomization analysis supported a causal relationship between NAFLD and cardio-cerebrovascular events. CONCLUSIONS: In patients with type 2 diabetes and chronic kidney disease, the NAFLD presence and elevated FIB-4 scores link to heightened mortality risk and ESKD susceptibility. Moreover, NAFLD shows a causal relationship with cardio-cerebrovascular events.

Investigation of indoleamine 2,3-dioxygenase 1 expression in uveal melanoma.

The purpose of this study was to investigate indoleamine 2,3-dioxygenase 1 (IDO1) expression and its implications in uveal melanoma (UM). Bioinformatics analysis was performed on microarray data (GSE22138 and GSE27831) from the Gene Expression Omnibus (GEO) database to evaluate IDO1 expression in mRNA level. Ninety-two cases in the database were divided into the IDO1-high group (46 cases) and IDO1-low group (46 cases). Paraffin embedded tumor sections from 27 patients with UM were studied by immunofluorescence. The mRNA results showed that IDO1 expression was inversely correlated with tumor thickness (9.93 ±â€¯3.33 mm in IDO1-high group vs. 11.56 ±â€¯2.38 mm in IDO1-low group) (p = 0.016) and metastatic rate (30.4% in IDO1-high group vs. 69.6% in IDO1-low group, p < 0.001). The IDO1-high group showed higher immune cell gene expression: CD3D (6.56 ±â€¯1.0 vs. 5.46 ±â€¯0.53, p < 0.0001), CD4 (4.72 ±â€¯0.4 vs. 4.2 ±â€¯0.42, p < 0.0001), and CD68 (6.17 ±â€¯1.23 vs. 5.53 ±â€¯0.77, p = 0.015). Further analysis showed that immune-suppressive T regulatory cell genes (CD3D, CD4, IL2RA and FOXP3) were expressed in 67.4% (31/46) cases in the IDO1-high group and 23.91% (11/46) cases in the IDO1-low group. In addition, IDO1 and interferon gamma (IFNG) mRNA expression were strongly correlated (r = 0.70, p < 0.0001). The correlation analysis of different immune checkpoints showed that IDO1 was positively correlated with CD274(PDL1), but not CTLA4 or PDCD1.The disease-free survival (DFS) in the IDO1-high/IFNG-high group was better than that of the IDO1-low/IFNG-low group. The IDO1 immunostaining result showed that 2 cases in 18 UMs with Bruch's membrane (BM) rupture and 7 out of 9 cases without BM rupture were scored high (Grade 2-3) (p = 0.001). Comparing the immune cells staining results between IDO1-high group and IDO1-low group, higher percentage of patients in the former group had high levels of T cells and macrophages infiltration, but only the difference in macrophage was statistically significant (CD68, 77.78% vs. 27.78%, p = 0.04). The analysis based on GEO data and the result from immunostaining study are consistent with each other. In conclusion, the expression of IDO1 is probably induced by IFNγ from infiltrated immune cells in UM. BM rupture is an important indicator of IDO1 expression level and distribution pattern. The complex role of IDO1 may limit its therapeutic effect in UM.

Association between lactate dehydrogenase and the risk of diabetic kidney disease in patients with type 2 diabetes.

Objective: This study aims to investigate the association between lactate dehydrogenase (LDH) and the risk of diabetic kidney disease (DKD) in patients with type 2 diabetes (T2D). Methods: The study enrolled patients with diagnosis of T2D between 2009 and 2018 from the National Nutrition and Health Examination Survey (NHANES) database. Demographic information, laboratory test, and diagnostic data were collected. Restricted cubic spline (RCS) plots were used to assess the dose-effect relationship between LDH levels and the risk of DKD in patients with T2D. Based on LDH levels, individuals were divided into higher and lower groups using dichotomy, and multivariate logistic regression analysis was conducted to explore the relationship between different LDH levels and the risk of DKD in T2D patients. Stratified analysis was performed to assess the consistency of the result. Results: A total of 4888 patients were included in the study, with 2976 (60.9%) patients without DKD and 1912 (39.1%) patients with DKD. RCS plots showed that the risk of DKD increased with increasing LDH levels. Multifactorial logistic regression analysis revealed that T2D patients with higher LDH levels had a 45% increased risk of DKD compared to those with lower LDH levels (OR=1.45; 95% CI: 1.11-1.89). Furthermore, each standard deviation increase in LDH level was associated with a 24% increase in DKD incidence among T2D patients (OR=1.24; 95% CI: 1.07-1.44). Stratified analysis consistently supported these findings. Conclusions: LDH can serve as a valuable biomarker for screening DKD in patients with T2D.

Febuxostat, a xanthine oxidase inhibitor, regulated long noncoding RNAs and protected the brain after intracerebral hemorrhage.

Intracerebral hemorrhage (ICH) is a devastating cerebrovascular disease and is associated with a high global health burden. Long noncoding RNAs are involved in the pathological damage of ICH. Febuxostat, one of the xanthine oxidase inhibitors, is commonly used in the treatment of hyperuricemia and has been studied in different pathological processes, and its protective effects have been proven in different organs. This study was conducted to investigate whether febuxostat protects brain via regulating long noncoding RNAs after ICH. The modified neurological severity score, wire hanging test, Evans blue perfusion and immunofluorescence were performed to test the protective effects of febuxostat in a mouse model of ICH. Whole transcriptome sequencing was conducted to identify the lncRNAs affected by febuxostat and their functions were analyzed. Febuxostat ameliorated behavioral abnormalities and protected the blood-brain barrier after ICH. Fifteen lncRNAs regulated by febuxostat after ICH were detected. These 15 lncRNAs were associated with 83 gene ontology items. In total, 35 genes, 15 mRNAs and 202 miRNAs were regarded as potential targets for the 15 lncRNAs; 183 co-expressed genes were identified for these 15 lncRNAs and the co-expression network was constructed. Potential binding between lncRNAs and mRNAs was also studied. Enrichment analysis revealed that the functions of the 15 lncRNAs were related to maintaining the blood-brain barrier. This study demonstrated febuxostat protected brain after ICH. Fifteen lncRNAs were regulated and were associated with the effects of febuxostat on BBB integrity after ICH.

Prognostic value of metabolic syndrome in renal structural changes in type 2 diabetes.

PURPOSE: To investigate the prognostic value of metabolic syndrome (MetS) and its relationship with renal structure changes in patients with type 2 diabetes and associated diabetic nephropathy (DN). METHODS: 411 Chinese patients with type 2 diabetes and biopsy-confirmed DN were enrolled in this retrospective study. MetS was defined according to the modified criteria of the 2005 International Diabetes Federation. Baseline demographics and clinical information at the time of renal biopsy were extracted from the hospital's electronic medical records system. Renal pathological findings were assessed according to Renal Pathology Society system. Univariate and multivariate logistic regression analyses were performed to define the pathological covariates associated with MetS. A competing risk model, with death as the competing risk, was used to estimate the sub-distribution hazard ratio (SHR) of MetS for end-stage kidney disease (ESKD). RESULTS: 224 (55%) patients had MetS. Patients with MetS had poor renal function and more severe interstitial fibrosis tubular atrophy scores (IFTA) than those without MetS. Multivariate logistic regression analysis revealed that IFTA was significantly associated with MetS (odds ratio per score increase 1.45, 95% confidence interval [CI] 1.02-2.05). Of the patients with DN at risk, 40% of patients progressed to ESKD. After adjusting for renal function and pathological parameters, the presence of MetS was an independent predictor for progression to ESKD (SHR 1.93, 95% CI 1.34-2.79). The SHRs for progression to ESKD also increased as the number of MetS components increased. Additionally, adding the IFTA scores improved the prognostic power of a model that only contained MetS and clinical covariates for predicting future ESKD. CONCLUSION: MetS is an independent prognostic predictor of ESKD in patients with T2D and DN, while adding the IFTA scores increased the prognostic value of MetS for renal outcome.

EDTA Promotes the Mineralization of Dental Pulp In Vitro and In Vivo.

INTRODUCTION: Dentin regeneration is one of the main goals of vital pulp treatment in which the biological properties of dental pulp cells (DPCs) need to be considered. In our previous study, we showed that EDTA could enhance the stromal cell-derived factor 1 alpha-induced migration of DPCs. The purpose of this study was to explore the effects of EDTA on the mineralization of dental pulp in vitro and in vivo. METHODS: DPCs were obtained from human premolars or third molars. Alkaline phosphatase assays and alizarin red S staining were used to examine the degree of differentiation and mineralized nodule formation of DPCs. Real-time polymerase chain reaction and Western blot analysis were performed to detect the messenger RNA and protein expressions of mineralization-related markers in DPCs. Extracellular-regulated protein kinase and Smad inhibitors were used to study the roles of these 2 signaling pathways in this process. In addition, pulp exposures were created on 18 premolars of 2 beagle dogs (>12 months) using a high-speed dental handpiece. The experimental group (n = 9) was treated with 12% EDTA for 5 minutes, and the control group (n = 9) was treated with sterile saline for the same duration. Mineral trioxide aggregate was used for direct pulp capping followed by glass ionomer cement sealing. Samples were collected 3 months later, and the regenerated dentin was assessed by micro-computed tomographic and histologic analyses. RESULTS: Exposure to 12% EDTA promoted the activity of alkaline phosphatase, the formation of mineralized nodules, and the messenger RNA and protein expressions of mineralization-related markers in DPCs. Furthermore, the process of 12% EDTA enhancing the differentiation of DPCs was mediated by the extracellular-regulated protein kinase 1/2 signaling pathway and inhibited by the Smad2/3 signaling pathway. In vivo, compared with the control group, more regenerated dentin that had fewer tunnel defects was formed in the 12% EDTA-treated group. CONCLUSIONS: Our results showed that 12% EDTA could promote the mineralization of dental pulp in vitro and in vivo.

Neuroprotection of Chikusetsu saponin V on transient focal cerebral ischemia/reperfusion and the underlying mechanism.

BACKGROUND: Oxidative stress and frequently unwanted alterations in mitochondrial structure and function are key aspects of the pathological cascade in transient focal cerebral ischemia. Chikusetsu saponin V (CHS V), a major component of saponins from Panax japonicas, can attenuate H2O2-induced oxidative stress in SH-SY5Y cells. PURPOSE: The aim of the present study was to investigate the neuroprotective effects and the possible underlying mechanism of CHS V on transient focal cerebral ischemia/reperfusion. METHODS: Mice with middle cerebral artery occlusion (MCAO) and cultured cortical neurons exposed to oxygen glucose deprivation (OGD) were used as in vivo and in vitro models of cerebral ischemia, respectively. The neurobehavioral scores, infarction volumes, H&E staining and some antioxidant levels in the brain were evaluated. The occurrence of neuronal death was estimated. Total and mitochondrial reactive oxygen species (ROS) levels, as well as mitochondrial potential were measured using flow cytometry analysis. Mitochondrial structure and respiratory activity were also examined. Protein levels were investigated by western blotting and immunohistochemistry. RESULTS: CHS V effectively attenuated cerebral ischemia/reperfusion (CI/R) injury, including improving neurological deficits, shrinking infarct volume and reducing the number of apoptotic cells. Furthermore, CHS V treatment remarkably increased antioxidant levels and reduced ROS levels and mitochondrial damage by enhancing the expression and deacetylation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by activating AMPK and SIRT-1, respectively. CONCLUSION: Our data demonstrated that CHS V prevented CI/R injury by suppressing oxidative stress and mitochondrial damage through the modulation of PGC-1α with AMPK and SIRT-1.

[A rapid histological preparation method for observation of morphology and composition distribution of tendon collagen fascicle and endotendinium].

OBJECTIVE: To explore a rapid histological preparation method to observe morphology and composition distribution of tendon collagen fascicle and endotendinum. METHODS: Taking porcine superflexor tendon of foot as an example, tendons were sliced into sections with 6 µm by frozen section technology, after which general observation of the section integrity was carried out. After fixed with 10% neutral buffered formalin and performed with HE staining, the tissue integrity and ice crystal formation were observed under microscope. Sections were then divided into 5 groups by different methods of dyeing. Group A: Priodic acid-Shiff (PAS) staining; group B: Masson staining; group C: reticular fibers staining; group D: immunohistochemical and immunofluorescent staining of type Ⅲ collagen; group E: the sections were baked at 65℃ for 10 minutes and stained with Masson. The composition distribution of tendon collagen fascicle and endotendinum in different groups were observed. RESULTS: From general observation, the frozen section of tendon tissue was complete and continuous. Although the tissue integrity in the tendon sections could be seen and no ice crystal was formed, the composition distribution could not be identified by HE staining. The entire tendons in groups A, B, and C were dyed, and the composition distribution of collagen fascicle and endotendinum could not be identified. The endotendinum in group D was stained weakly positive for type Ⅲ collagen alone, and the two components were differentiated dyed but the contrast was not obvious. In group E, the collagen fascicle and endotendinium were differentiated dyed and the two components in tendon tissue were clearly visible. CONCLUSION: The morphology and the composition distribution of tendon collagen fascicle and endotendinum can be characterized rapidly and accurately, using a combination of baking at 65℃ for 10 minutes and Masson staining after porcine superflexor tendons were sliced by frozen section technology.