Background: Although the gut microbiota is involved in metabolic disease such as atherosclerosis, the underlying mechanism remains elusive. Paeonol (Pae) is a natural phenolic compound isolated from Cortex Moutan, which exhibits anti-atherosclerotic effects. Our previous research demonstrated gut microbiota as a site of Pae action. However, the mechanism by which Pae exerts its anti-atherosclerotic effect by the regulation of gut microbiota remains unclear.Objective: To investigate a potential mechanistic link between the gut microbial lipopolysaccharide (LPS) and vascular smooth muscle cell (VSMC) proliferation in atherosclerosis progression and explore the possible role of Pae.Methods: Experimental atherosclerosis was established in ApoE−/− mice, and the atherosclerosis mice were treated with Pae for 4 weeks before being sacrificed for analyses while conducting fecal microbiota transplantation (FMT). The plaque area, levels of serum LPS, expressions of inflammatory factors in serum or aorta, and intestinal barrier permeability were determined. VSMCs were co-cultured with THP-1 cells. CCK-8 assay and EdU staining were performed to assess the proliferative capacity of VSMCs. Immunofluorescence staining was performed to observe the nuclear transfer of p65. Western blotting was used to detect the candidate protein expression level, and quantitative real-time PCR (qRT-PCR) was used to detect the mRNA expression level in tissues or cells of each group.Results: During atherosclerosis progression, gut dysbiosis leads to the peripheral accumulation of gut microbial LPS, which acts as a trigger to stimulate osteopontin (OPN) production from circulating monocytes, inducing cell-to-cell crosstalk to promote VSMC proliferation in the aorta. Importantly, the elevation of LPS and OPN concentrations in the blood was also observed in patients with atherosclerosis. Pae could significantly improve atherosclerosis, suppress gut microbial LPS accumulation, and inhibit monocyte/macrophage activation and VSMC proliferation.Conclusions: The present study provides a mechanistic scenario for how long-term stimulation of gut microbial LPS in circulating blood generates a pathological secondary response that leads to abnormal proliferation of VSMCs using high OPN expression in circulating monocytes and suggests a novel strategy for atherosclerosis therapy by remodeling the gut microbiota.

BackgroundConventionally, serum ceruloplasmin levels below the lower reference limit (0. 20 g/L) is considered a diagnostic cutoff point for Wilson's disease (WD). However, the lower reference limit varies with assay methodologies and the individuals in the included studies. The objective of this study was to determine the optimal cutoff value of serum ceruloplasmin levels for the diagnosis of WD in a large Chinese cohort and to identify factors associated with serum ceruloplasmin.MethodsThe cutoff value of ceruloplasmin levels was developed based on a retrospective derivation cohort of 3,548 subjects (1,278 patients with WD and 2,270 controls) and was validated in a separate validation cohort of 313 subjects (203 patients with WD and 110 controls). The performance of immunoassay was tested by receiver operating characteristic curve (ROC) analysis, and differences among the groups were analyzed by using the Mann–Whitney U-test and the Kruskal–Wallis test.ResultsThe conventional cutoff of serum ceruloplasmin levels of <0.2 g/L had an accuracy of 81.9%, which led to a false-positive rate of 30.5%. The optimal cutoff of the serum ceruloplasmin level for separating patients with WD from other participants was 0.13 g/L, as determined by ROC analysis. This cutoff value had the highest AUC value (0.99), a sensitivity of 97.0%, and a specificity of 96.1%. Moreover, it prevented unnecessary further investigations and treatments for 492 false-positive patients. By determining the correlation between serum ceruloplasmin and phenotypes/genotypes in patients with WD, we found that the serum ceruloplasmin level was lower in early-onset patients and higher in late-onset patients. Interestingly, patients with the R778L/R919G genotype had higher serum ceruloplasmin levels than patients with other hot spot mutation combinations.ConclusionOur work determined the optimal cutoff value of serum ceruloplasmin levels for the diagnosis of WD and identified differences in serum ceruloplasmin levels with respect to the age of symptom onset and ATP7B mutations, which may provide some valuable insights into the diagnosis and counsel of patients with WD.

Background: Paeonol (Pae)， the main active compound of the root of Paeonia albiflora, is efficacious in treating atherosclerosis (AS). Endothelial dysfunction is throughout the pathological progression of AS. It is expected that inhibition of Endothelial-to-mesenchymal transition (EndMT) will be a key target for AS treatment. Objective: In this study, we investigated the molecular mechanism of the regulatory effect of Pae on EndMT in human umbilical vein endothelial cells (HUVECs). Methods: Cell cytotoxicity, proliferation, and migration were detected by CCK-8, the wound healing assay, and EdU staining, respectively. The protein expressions were measured by Western blot or immunofluorescence staining. Immunofluorescence staining was performed to indicate endothelial cells undergoing EndMT in ApoE-/- mice. In vitro TGF-β1-induced EndMT assays were performed in HUVECs and the effect of Pae was explored. Results: We demonstrated that Pae could improve induced TGF-β1-EndMT in vivo and in vitro. Mechanism study revealed that Pae directly bonds to the activin-like kinase 5 (ALK5, also known as TGFβ type I receptor), inhibited downstream Smad2/3 phosphorylation, and thus alleviated EndMT. Notably, overexpression of ALK5 significantly reversed the inhibitory effect of Pae on EndMT in HUVECs. Conclusion: Our results indicate that ALK5 is a promising druggable target for AS, and pharmacological regulation of ALK5-Smad2/3 signaling pathway with small-molecule holds great potential to benefit AS patients.

Background In Wilson’s disease (WD) patients, network connections across the brain are disrupted, affecting multidomain function. While the details of this neuropathophysiological mechanism remain unclear due to the rarity of WD. In this study, we aimed to investigate alterations in brain network connectivity at the whole-brain level (both intra- and inter-network) in WD patients through independent component analysis (ICA) and the relationship between alterations in these brain network functional connections (FCs) and clinical neuropsychiatric features to understand the underlying pathophysiological and central compensatory mechanisms.Methods 85 patients with WD and age- and sex-matched 85 healthy control (HC) were recruited for resting-state functional magnetic resonance imaging (rs-fMRI) scanning. We extracted the resting-state networks (RSNs) using the ICA method, analyzed the changes of FC in these networks and the correlation between alterations in FCs and clinical neuropsychiatric features.Results Compared with HC, WD showed widespread lower connectivity within RSNs, involving default mode network (DMN), frontoparietal network (FPN), somatomotor network (SMN), dorsal attention network (DAN). Furthermore, the decreased FCs in the left medial prefrontal cortex (L_ MPFC), left anterior cingulate gyrus (L_ACC), precuneus (PCUN)within DMN were negatively correlated with the Unified Wilson’s Disease Rating Scale-neurological characteristic examination (UWDRS-N), and the decreased FCs in the L_MPFC, PCUN within DMN were negatively correlated with the Unified Wilson’s Disease Rating Scale-psychiatric symptoms examination (UWDRS-P). We additionally discovered that the patients with WD exhibited significantly stronger FC between the FPN and DMN, between the DAN and DMN, and between the FPN and DAN compared to HC.Conclusions we have provided evidence that WD is a disease with widespread dysfunctional connectivity in resting networks in brain, leading to neurological features and psychiatric symptoms (e.g. higher-order cognitive control and motor control impairments). The altered intra- and inter-network in the brain may be the neural underpinnings for the neuropathological symptoms and the process of injury compensation in WD patients.

Dysfunction of the lenticular nucleus is thought to contribute to neurological symptoms in Wilson’s disease (WD). However, very little is known about whether and how the lenticular nucleus influences dystonia by interacting with the cerebral cortex and cerebellum. To solve this problem, we recruited 37 WD patients (20 men; age, 23.95 ± 6.95 years; age range, 12–37 years) and 37 age- and sex-matched healthy controls (HCs) (25 men; age, 25.19 ± 1.88 years; age range, 20–30 years), and each subject underwent resting-state functional magnetic resonance imaging (RS-fMRI) scans. The muscle biomechanical parameters and Unified Wilson Disease Rating Scale (UWDRS) were used to evaluate the level of dystonia and clinical representations, respectively. The lenticular nucleus, including the putamen and globus pallidus, was divided into 12 subregions according to dorsal, ventral, anterior and posterior localization and seed-based functional connectivity (FC) was calculated for each subregion. The relationships between FC changes in the lenticular nucleus with muscle tension levels and clinical representations were further investigated by correlation analysis. Dystonia was diagnosed by comparing all WD muscle biomechanical parameters with healthy controls (HCs). Compared with HCs, FC decreased from all subregions in the putamen except the right ventral posterior part to the middle cingulate cortex (MCC) and decreased FC of all subregions in the putamen except the left ventral anterior part to the cerebellum was observed in patients with WD. Patients with WD also showed decreased FC of the left globus pallidus primarily distributed in the MCC and cerebellum and illustrated decreased FC from the right globus pallidus to the cerebellum. FC from the putamen to the MCC was significantly correlated with psychiatric symptoms. FC from the putamen to the cerebellum was significantly correlated with muscle tension and neurological symptoms. Additionally, the FC from the globus pallidus to the cerebellum was also associated with muscle tension. Together, these findings highlight that lenticular nucleus–cerebellum circuits may serve as neural biomarkers of dystonia and provide implications for the neural mechanisms underlying dystonia in WD.

Background: Neuroimaging studies in Wilson’s disease (WD) have identified various alterations in white matter (WM) microstructural organization. However, it remains unclear whether these alterations are localized to specific regions of fiber tracts, and what diagnostic value they might have. The purpose of this study is to explore the spatial profile of WM abnormalities along defined fiber tracts in WD and its clinical relevance. Methods: Ninety-nine patients with WD (62 men and 37 women) and 91 age- and sex-matched controls (59 men and 32 women) were recruited to take part in experiments of diffusion-weighted imaging with 64 gradient vectors. The data were calculated by FMRIB Software Library (FSL) software and Automated Fiber Quantification (AFQ) software. After registration, patient groups and normal groups were compared by Mann–Whitney U test analysis. Results: Compared with the controls, the patients with WD showed widespread fractional anisotropy reduction and mean diffusivity, radial diffusivity elevation of identified fiber tracts. Significant correlations between diffusion tensor imaging (DTI) parameters and the neurological Unified Wilson’s Disease Rating Scale (UWDRS-N), serum ceruloplasmin, and 24-h urinary copper excretion were found. Conclusions: The present study has provided evidence that the metrics of DTI could be utilized as a potential biomarker of neuropathological symptoms in WD. Damage to the microstructure of callosum forceps and corticospinal tract may be involved in the pathophysiological process of neurological symptoms in WD patients, such as gait and balance disturbances, involuntary movements, dysphagia, and autonomic dysfunction.

Gandouling (GDL) Pill is a novel Traditional Chinese medicinal drug to treat Wilson’s disease in clinics. It is composed of six separate herbal medicines, including Rhei Radix ET Rhizoma, Coptidis Rhizoma, Salviae Miltiorrhizae Radix ET Rhizoma, Spatholobi Caulis, Curcumae Rhizoma, and Curcumae Longae Rhizoma. In this study, a strategy was proposed to investigate the chemical constituents and to quantify the potential bioactive components in GDL Pill. Firstly, the mass fragmentation behaviors of representative compounds were investigated, and, in total, 69 compounds were characterized in GDL Pill using full scan/dd-MS2 scan mode by ultra-high-performance liquid chromatography (UPLC)/Q-Orbitrap mass spectrometry (MS). These compounds included 18 alkaloids, 18 ketones, 16 phenolic compounds, 11 organic acids, and 6 tanshinones. Seventeen of the compounds were unambiguously identified by comparison with reference standards. Secondly, the absorption components of GDL Pill in rat plasma were investigated by using target-Selected Ion Monitoring (t-SIM) scan mode built in Q-Orbitrap MS. A total of 18 components were detected, which were considered as potential bioactive components of GDL Pill. Thirdly, 10 major absorption components were simultaneously determined in six batches of samples by UPLC/diode array detector (DAD). The method was fully validated with respect to linearity, precision, repeatability, stability, and recovery. Alkaloids from Coptidis Rhizoma, such as coptisine (8), berberine (18), palmatine (19), were the most abundant bioactive compounds for GDL Pill that possess the potential be used as quality markers. The proposed strategy is practical and efficient for revealing the material basis of GDL Pill, and also provides a simple and accurate method for quality control.