Molecular Medicine最新文献

Background: Dementia with Lewy bodies (DLB) is the second most common type of degenerative dementia in older patients. As with other multifactorial diseases, the pathogenesis results from interactions of environmental and genetic factors. The genetic basis of DLB is not yet fully understood. Recent genomic analyses of DLB in Caucasian cohorts identified genetic susceptibility loci for DLB, but the comprehensive genomic analysis in Asians was still not performed.

Methods: We conducted a genome-wide association study (GWAS) in Japanese subjects (211 DLB cases and 6113 controls) to clarify the genetic architecture of DLB pathogenesis.

Results: We identified the East Asian-specific DHTKD1 locus (rs138587229) on chromosome 10 with genome-wide significance (GWS; P = 3.27 $\times$ 10^-8) and the ICOS/PARD3B locus on chromosome 2 with suggestive significance (P = 3.95 $\times$ 10^-7) as novel DLB genetic risk loci. We also confirmed the APOE locus (rs429358, P < 5.0 × 10^-8), a known risk locus for DLB and Alzheimer's disease in Caucasians. The DHTKD1 locus was associated with the gene expression of SEC61A2 and showed a causal relationship with cholinesterase levels. In a trans-ethnic meta-analysis that included Japanese, UK Biobank, and other Caucasian GWAS, we confirmed the risk for DLB at APOE and SNCA loci with GWS. Transcriptome-wide association analysis identified ZNF155 and ZNF284 in the brain cortex and GPRIN3 in the substantia nigra as putative causal genes for DLB.

Conclusions: This is the first GWAS for DLB in East Asians, and our findings provide new biological and clinical insights into the pathogenesis of DLB.

Background: Although randomized clinical trials revealed that inhibitors of sodium-glucose cotransporter 2 (SGLT2) reduced the risk of cardiovascular and renal events in patients with type 2 diabetes, the underlying molecular mechanisms remain to be elucidated. Since there is accumulating evidence that AGEs and their receptor (RAGE) play a crucial role in diabetes-related complications, we examined here whether empagliflozin ameliorates renal and metabolic derangements in db/db mice, an obese type 2 diabetes animal by blocking the AGE-RAGE axis.

Methods: Eight-week-old db/db mice were fed a 0.045% empagliflozin diet (db/db + Empa) or normal diet (db/db) for 13 weeks. The same week-old db/ + m mice were maintained on normal diet (db/ + m) used as a control. At baseline and 13 weeks after intervention, biochemical analyses in the serum and urine were performed, and kidneys and adipose tissues were obtained for morphological, immunohistochemical, and reverse transcription-polymerase chain reaction analyses.

Results: Empagliflozin treatment for 13 weeks significantly reduced AGEs, N^ε-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), RAGE, NADPH oxidase-derived oxidative stress, inflammatory and fibrotic reactions in the kidneys of db/db mice of 21-week-old in association with attenuation of glomerular extracellular matrix accumulation, podocyte loss, proteinuria, and tubulointerstitial damage. Empagliflozin also reduced the AGE-RAGE-oxidative stress-induced inflammatory reactions in the adipose tissues of db/db mice, which was associated with restoration of adiponectin levels and decreased insulin resistance. Serum MG-H1 levels of control and db/db mice at 21 weeks of age were significantly associated with proteinuria, tubulointerstitial damage, tissue AGEs levels, and serum monocyte chemoattractant protein-1 and adiponectin (inversely) values.

Conclusions: Our present findings suggest that empagliflozin could ameliorate renal and metabolic derangements in type 2 diabetes animals by attenuating the AGE-RAGE axis.

Background: MicroRNAs (miRNAs) regulate biological processes by inhibiting translation and causing mRNA degradation. In this study, we identified the miRNAs involved in the development and progression of lupus nephritis (LNs) and verified their roles.

Methods: Total RNA, extracted from PBMCs collected from patients with LNs before and after treatment, was used for miRNA array analysis to identify miRNAs whose expression was significantly altered. The results of this analysis were confirmed using qRT-PCR. The identified miRNAs were transfected into normal human mesangial cells (NHMCs), human renal proximal tubule epithelial cells (RPTECs), human umbilical vein endothelial cells (HUVECs), and THP-1-derived macrophages (THP1-Mφ) to investigate their biological functions.

Results: Three miRNAs were altered in PBMCs before and after treatment of LNs. Among these miRNAs, hsa-miR-6516-3p promoted TNF-α-induced expression of MMP-9 in NHMCs. Moreover, hsa-miR-6516-3p downregulated the expression of RECK, an endogenous inhibitor of MMP-9. However, in NHMCs, endogenous hsa-miR-6516-3p was not present in functional amounts under inflammatory environment; therefore, we performed analysis using an experimental system considering extracellular influences of mesangial cells under LNs. The expression of hsa-miR-6516-3p was increased in HUVECs under inflammatory conditions and in activated macrophages.

Conclusions: hsa-miR-6516-3p increases MMP9 expression by suppressing RECK, and might, thereby, exacerbate LNs.

Background: Cancer stem cells are a small subpopulation of cells which are responsible for tumor metastasis, angiogenesis, drug resistance etc. 5-Fluorouracil (5FU), a common therapeutic drug used in colorectal cancer treatment is reported to enrich CSCs, tumor recurrence and induces severe organ toxicities resulting in poor clinical outcome in patients. Therefore, we introduced a natural flavonoid Orientin in combination with 5FU to mitigate the CSC mediated angiogenesis and induced toxicities.

Methods: Tumorosphere generation, flow cytometry, immunofluorescence assay, and western blotting were performed by using 5FU and Orientin individually and both treated colorectal cells and CSCs. In silico study was carried out to check the interaction between HIF1α and Orientin. In ovo chorioallantoic membrane (CAM) assay and tube formation assay using HUVECs were performed to monitor CSC mediated angiogenesis. In vivo CT26 syngeneic mice model was used to validate in silico and ex vivo results.

Results: We found that 5FU treatment significantly increased the CD44⁺/CD133⁺ CSC population. In contrast, this CSC population in CSC enriched spheres (CES) derived from HCT116 cells were decreased by combination of Orientin and 5FU. Decrease of CSC's stemness properties was also noted, as evidenced by the downregulation of NANOG, SOX2 and OCT4. This new therapeutic strategy also inhibited CSC mediated angiogenesis by downregulating 5FU induced ROS, NO and LPO in those tumorospheres. Combination of Orientin and 5FU significantly reduced CSC mediated angiogenesis in HUVEC and CAM. Additionally, in silico study predicted that Orientin can bind to the PAS domain of HIF1α, a crucial factor for promoting angiogenesis. Expression of HIF1α and VEGFA were also decreased when the CESs were exposed to the combinatorial treatment. Additionally, we found that treatment with 5FU alone resulted reduction in tumor volume but it enriched CSCs and produced nephrotoxicity and hepatotoxicity in vivo. Combined treatment also considerably reduced the CD44⁺/CD133⁺ CSC population and hindered angiogenesis in a therapeutic in vivo model in BALB/c mice.

Conclusions: This novel treatment strategy of "Orientin with 5FU" is likely to improve the efficiency of conventional chemotherapy and may suppress disease recurrence in colorectal cancer by limiting CSC mediated angiogenesis.

The regulatory mechanisms by which cholesterol influences hair regeneration remain incompletely understood. This study investigates the effects of cholesterol on hair follicle stem cells (HFSCs) proliferation and hair regeneration, with a focus on the underlying molecular mechanisms. Subcutaneous cholesterol injections in C57BL/6 mice significantly enhanced hair regeneration by promoting HFSCs proliferation. Hematoxylin and eosin (HE) staining revealed a greater number of hair follicles in the anagen phase in the cholesterol-treated group compared to controls. Immunofluorescence (IF) and BrdU labeling further confirmed that cholesterol significantly stimulated HFSCs proliferation. Mechanistically, cholesterol activated the PKA signaling pathway, leading to the phosphorylation of tyrosine hydroxylase (TH) at the serine 40 residue, which subsequently stimulated the sympathetic nervous system (SNS). SNS activation enhanced HFSCs proliferation and increased the proportion of hair follicles in the anagen phase. Furthermore, sympathetic nerve ablation significantly attenuated the hair regeneration-promoting effects of cholesterol, highlighting the critical regulatory role of SNS in this process. These findings provide key insights into the molecular mechanisms by which cholesterol regulates hair regeneration via the PKA-tyrosine hydroxylase-SNS pathway. Moreover, they suggest potential therapeutic applications targeting cholesterol-mediated signaling pathways to promote hair regeneration.

As an emerging technology, organoids are promising new tools for basic and translational research in disease. Currently, the culture of organoids relies mainly on a type of unknown composition scaffold, namely Matrigel, which may pose problems in studying the effect of mechanical properties on organoids. Hydrogels, a new material with adjustable mechanical properties, can adapt to current studies. In this review, we summarized the synthesis of recent advance in developing definite hydrogel scaffolds for organoid culture and identified the critical parameters for regulating mechanical properties. In addition, classified by different mechanical properties like stiffness and viscoelasticity, we concluded the effect of mechanical properties on the development of organoids and tumor organoids. We hope this review enhances the understanding of the development of organoids by hydrogels and provides more practical approaches to investigating them.

Background and purpose: Shwachman-Diamond Syndrome (SDS) is an autosomal recessive disease belonging to the inherited bone marrow failure syndromes and characterized by hypocellular bone marrow, exocrine pancreatic insufficiency, and skeletal abnormalities. SDS is associated with increased risk of developing myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). Although SDS is not primarily considered an inflammatory disorder, some of the associated conditions (e.g., neutropenia, pancreatitis and bone marrow dysfunction) may involve inflammation or immune system dysfunctions. We have already demonstrated that signal transducer and activator of transcription (STAT)-3 and mammalian target of rapamycin (mTOR) were hyperactivated and associated with elevated IL-6 levels in SDS leukocytes. In this study, we analyzed the level of phosphoproteins involved in STAT3 and mTOR pathways in SDS lymphoblastoid cells (LCLs) and the secretomic profile of soluble pro-inflammatory mediators in SDS plasma and LCLs in order to investigate the systemic inflammation in these patients and relative pathways.

Methods: Twenty-six SDS patients and seven healthy donors of comparable age were recruited during the programmed follow-up visits for clinical evaluation at the Verona Cystic Fibrosis Center Human. The obtained samples (plasma and/or LCLs) were analyzed for: phosphoproteins, cytokines, chemokines and growth factors levels by Bio-plex technology; microRNAs profiling by next generation sequencing (NGS) and microRNAs expression validation by Real Time-PCR (RT-PCR) and droplet digital PCR (ddPCR) .

Results: We demonstrated dysregulation of ERK1/2 and AKT phosphoproteins in SDS, as their involvement in the hyperactivation of the STAT3 and mTOR pathways confirmed the interplay of these pathways in SDS pathophysiology. However, both these signaling pathways are strongly influenced by the inflammatory environment. Here, we reported that SDS is characterized by elevated plasma levels of several soluble proinflammatory mediators. In vitro experiments show that these pro-inflammatory genes are closely correlated with STAT3/mTOR pathway activation. In addition, we found that miR-181a-3p is down-regulated in SDS. Since this miRNA acts as a regulator of several pro-inflammatory pathways such as STAT3 and ERK1/2, its down-regulation may be a driver of the constitutive inflammation observed in SDS patients.

Conclusions: The results obtained in this study shed light on the complex pathogenetic mechanism underlying bone marrow failure and leukemogenesis in SDS, suggesting the need for anti-inflammatory therapies for SDS patients.

Background: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. Protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification has been found to affect the processing of several important proteins implicated in ALS. However, the overall level and cellular localization of O-GlcNAc during ALS progression are incompletely understood, and large-scale profiling of O-GlcNAcylation sites in this context remains unexplored.

Methods: By using immunostaining analysis and chemoenzymatic labeling-based quantitative chemoproteomics, we assayed O-GlcNAcylation dynamics of lumbar spinal cords from SOD-G93A mice and their non-transgenic (NTG) littermates, the most widely used animal model for studying ALS pathogenesis.

Results: We discovered that the global O-GlcNAcylation was significantly reduced at the disease end stage. Correlatively, a great increase of OGA was observed. Immunohistochemistry and immunofluorescence analysis showed a higher proportion of O-GlcNAc-positive neurons in the NTG group, while O-GlcNAc colocalization with astrocytes/microglia was elevated in SOD1-G93A mice. Moreover, we reported the identification of 568 high-confidence O-GlcNAc sites from end-stage SOD1-G93A and NTG mice. Of the 568 sites, 226-many of which occurred on neuronal function and structure-related proteins-were found to be dynamically regulated.

Conclusion: These data provide a valuable resource for dissecting the functional role of O-GlcNAcylation in ALS and shed light on promising therapeutic avenues for ALS. The chemoenzymatic labeling-based chemoproteomic approach is applicable for probing O-GlcNAc dynamics in various pathological processes.

Myocardial ischemia/reperfusion (I/R) injury is a major global health problem with high rates of mortality and disability, which is more severe in patients with diabetes. Substantial researches have documented that diabetic myocardium are more susceptible to I/R injury, but many current intervention strategies against myocardial I/R injury have limited effectiveness in diabetic hearts. Caveolin-3 (Cav-3) is the signature protein of caveolae and serves as a signal integration and transduction platform in the plasma membrane of cardiomyocytes, which plays a vital role in myocardial functions, metabolism and protection of multiple conditioning strategies against I/R injury. Nevertheless, numerous studies have revealed that the expression of Cav-3 is impaired in diabetic hearts, which contributes to increased vulnerability of myocardium to I/R injury and resistance to protective conditioning strategies. In this review, we outline the basic structure and function of Cav-3, emphatically present the unique role of Cav-3 as a signal integration and transduction element in diabetic myocardial I/R injury and discuss its therapeutic perspective in strategies against myocardial I/R injury in diabetes.

Purpose: The ultra-high dose rate (UHDR) radiation shows promise in eradicating tumors while reducing normal tissue toxicities. However, the biological outcomes of UHDR are influenced by various factors, particularly the mean dose rate and instantaneous dose rate. Additionally, the UHDR response at large field sizes is lacking. This study aimed to explore the impact of different dose rate combinations on gastrointestinal biological outcomes following total-body irradiations (TBI) and to examine the involved molecular signaling pathways.

Method: Female C57BL6/J mice received 10 Gy TBI using three modes: ultra-high mean and ultra-high instantaneous dose rate irradiation (HH mode), low mean and ultra-high instantaneous dose rate irradiation (LH mode), and low mean and low instantaneous dose rate irradiation (LL mode). Mice were euthanized at 3 h and 48 h post irradiation to assess acute normal tissue damage and perform transcriptome sequencing. Furthermore, a subset of mice was monitored for 30 days to evaluate survival.

Results: We found that when the instantaneous dose rate is sufficiently high (> 10⁵ Gy/s), both ultra-high or low mean dose rate irradiation reduced mice mortality, myelosuppression, DNA damage, and cell apoptosis. The survival probabilities 30 days after 10 Gy TBI were 4/7, 4/6, and 0/6 in the HH, LH, and LL groups, respectively. Myelosuppression was lower at 3 h and 48 h post HH and LH irradiations than LL irradiation. The better regulated inflammatory response was evident at 48 h post HH and LH irradiation compared to LL irradiation. Additionally, DNA damages and cell apoptosis in the intestinal tissue were significantly reduced after HH and LH irradiations compared to LL irradiation. Transcriptome sequencing of intestinal tissues revealed that HH irradiation activated immune response pathways and suppressed mitochondrial related pathways compared to LL irradiation.

Conclusion: Our findings underscore the pivotal role of instantaneous dose rate in reducing radiation damages. When the instantaneous dose rate is sufficiently high (> 10⁵ Gy/s), both ultra-high or low mean dose rate irradiation (HH and LH mode) reduced mice mortality, myelosuppression, DNA damage, and cell apoptosis. Understanding these dose rate effects and biological responses are crucial for optimizing radiotherapy strategies and exploring the potential benefits of UHDR irradiation.