Supplementary Material for: Kaempferol ameliorated alcoholic hepatitis through improving intestinal barrier function by targeting miRNA-155 signaling

Introduction: To investigate the effect and mechanism of Kaempferol on alcoholic steatohepatitis. Methods: C57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. Kaempferol was given as the interventional drug to chronic alcohol-fed mice for six weeks to assess its effects. In vitro, intestinal epithelial Caco-2 cells were stimulated by alcohol, and miRNA-155 mimics were used to further study the effect of kaempferol to miRNA-155 signaling in intestinal epithelial cells. HE staining and oil red O staining were used to observe the liver and intestinal tissue damage in each group of mice, and ALT, AST, IL-1B and TNF-a were detected by kits; LPS expression was detected by ELISA kit, and the expression of IL-1B and TNF-a was assessed by qRT-PCR; activated inflammatory response of liver and colon tissue and the related signalling pathway activation. Results: Kaempferol treatment significantly improved pathological changes such as steatosis and vacuolated lesions in liver tissue of the alcohol diet model group, and reduced serum ALT and AST enzyme activities and liver tissue interleukin-1β and tumour necrosis factor-α mRNA expression levels. Kaempferol significantly reduced the expression of miRNA-155 in the intestinal tissue of alcohol-fed mice, significantly increased their SOCS1 protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and ZO-1. What's more, kaempferol significantly reduced serum LPS levels in ASH mice. In vitro experiments showed that compared with the control group, kaempferol significantly inhibited the expression level of miRNA-155 in Caco-2 cells under ethanol exposure, decreased the activation of nuclear factor kappa-B, led to an increase in the expression of SOCS1 protein, and increased the production level of occludin protein in Caco-2 cells under the effect of alcohol. In contrast, overexpression of miRNA-155 significantly decreased occludin and SOCS1 protein production and increased nuclear factor kappa-B activation levels in Caco-2 cells, and the administration of kaempferol significantly inhibited this effect. Conclusion: Kaempferol improved the stability of gut barrier function to ameliorate hepatic injury induced by alcohol intake through enhancing occludin protein expression, by targeting miR-155 to inhibit the excessive inflammatory response in the intestine

Supplementary Material for: Physiological Signatures of Dual Embryonic Origins in Mouse Skull Vault

<b><i>Background/Aims:</i></b> The mammalian skull vault is a highly regulated structure and consists of several membrane bones of different tissue origins (e.g. neural crest derived frontal bone and mesoderm derived parietal bone). Although membrane bones form through intramembranous ossification, neural crest derived frontal bone has superior osteoblast activity and bone regeneration ability, triggering a novel conception for craniofacial reconstruction and bone regeneration called endogenous calvarial regeneration. However, a comprehensive landscape of the genes and signaling pathways involved in this process is not clear. <b><i>Methods:</i></b> Transcriptome analysis within the two bone elements is firstly performed to determine the physiological signatures of differential gene expressions in mouse skull vault. <b><i>Results:</i></b> Frontal bone tissues and parietal bone tissues maintain tissue origin through special gene expression similar to neural crest vs mesoderm tissue, and physiological functions between these two tissues are also found in differences related to proliferation, differentiation and extracellular matrix production and clustered signaling pathways. <b><i>Conclusion:</i></b> Our data provide novel insights into the potential gene regulatory network in regulating the development of neural crest-derived frontal bone and mesoderm-derived parietal bone

Supplementary Material for: Demineralized Dentin Matrix Induces Odontoblastic Differentiation of Dental Pulp Stem Cells

<br>The aim of this study was to investigate the effect of demineralized dentin matrix (DDM) on dental pulp stem cells (DPSCs) and the potential of complexes with DPSCs and DDM for mineralized tissue formation. Stem cells derived from the dental pulp of healthy pigs aged 18 months were isolated and cultured. DPSCs were incubated with alpha-minimum essential medium treated with DDM extract at 1 mg/ml (DDM1) or 10 mg/ml (DDM10). The concentrations of 3 growth factors in DDM extract was measured by enzyme-linked immunosorbent assay. Adhesion of DPSCs on DDM and hydroxyapatite-tricalcium phosphate (HA-TCP) surfaces was observed using scanning electron microscopy. Cell proliferation was evaluated with cell counting kit-8 and migration by Transwell migration assays. Odontoblastic differentiation was assessed by alkaline phosphatase (ALP) and alizarin red staining, ALP activity and real-time polymerase chain reaction analysis of markers of ALP, runt-related transcription factor 2, type I collagen, dentin matrix acidic phosphoprotein-1, osteonectin and dentin sialophosphoprotein (DSPP). Finally, DPSCs were combined with DDM and placed subcutaneously in nude mice for 12 weeks; DPSCs combined with HA-TCP and DDM alone served as controls. DDM could promote DPSC adhesion, migration and odontoblastic differentiation. Mineralized tissue formation was observed with the DPSC and DDM combination and the DPSC and HA-TCP combination. The mineralized tissue of the DPSC + DDM combination stained positive for DSPP, similar to the dentin tissue. These results indicate that DDM induces DPSC odontoblastic differentiation, suggesting applications for dentin regeneration

Supplementary Material for: CD4+ T Lymphocytes, Especially Th2 Cells, Contribute to the Progress of Renal Fibrosis

<b><i>Background:</i></b> Renal tubulointerstitial fibrosis is the final common stage of renal failure. CD4+ T lymphocyte recruitment and activation after injury could be the very important early event that mediates the onset of renal fibrogenesis. But the role of CD4+ T lymphocytes in renal fibrosis is controversial and its cellular mechanism needs to be further investigated. <b><i>Methods:</i></b> Biopsy specimens were from patients with minimal-change or IgA nephropathy. Mouse renal fibrosis was induced by unilateral ureteral obstruction (UUO). CD4+ T lymphocytes of wild BALB/c mice were depleted with anti-CD4 antibody. BALB/c Nu/Nu mice were reconstituted with polarized Th1 or Th2 cells by tail vein injection. <b><i>Results:</i></b> Our study demonstrated that massive CD4+ T lymphocytes infiltrated fibrotic kidneys of patients. The depletion of CD4+ T lymphocytes inhibited UUO-induced mouse renal fibrosis. In the process of UUO-induced renal fibrosis, the ratios of Th2/Th1 increased with time. Results have also shown that Th2-reconstituted mice developed renal fibrosis more easily than Th1-reconstituted mice, which manifested by interstitial expansion and collagen deposition, higher expression of α-SMA and vimentin and increased expression of fibronectin, TGF-β and collagen I. We also found that CD4+ T cells from Th1-reconstitued mice tended to secrete IL-4 and IL-13 Th2-like cytokines. <b><i>Conclusion:</i></b> In conclusion, our study demonstrated the importance of CD4+ T lymphocytes in renal fibrosis and gave the first direct evidence that Th2 cells play a pivotal role in UUO-induced renal fibrosis. Inhibition of CD4+ T lymphocyte differentiation to Th2 would be a potential therapeutic intervention to prevent renal fibrosis

Supplementary Material for: The Characteristics and Treatment Outcomes of 71 Duodenal Brunner’s gland Adenomas with Endoscopic Submucosal Dissection

Abstract Background: The aim of this study was to investigate outcomes of patients with duodenal Brunner’s gland adenomas (BGAs) that were treated endoscopically. Methods: We identified consecutive 71 patients treated at our center with endoscopic submucosal dissection (ESD) for their duodenal tumors diagnosed pathologically as BGAs over the period between January 1, 2011 and December 31, 2021. We retrospectively analyzed our experience and short- and long-term outcomes of ESD therapy on patients with BGAs. Results: Among 71 BGA patients with an average age of 57 ± 11.7 years (range: 30-82), 48 (67.6%) were male and 23 (32.4%) were female. The accuracy of preoperative diagnosis with endoscopic ultrasonography was 44.0% (22/50). The H. pylori infection was found in 29 patients (29/71, 40.8%). The median size of BGAs was 1.5 cm [interquartile range (IQR) 0.8-2.7cm]. The most common location was the duodenum bulb (50/71, 64.8%). For the ESD procedure, the median operation time was 15.0 minutes (IQR 9.5-25.5 minutes). The en-bloc and the complete resection rates were 97.2% and 92.3%, respectively. ESD-related mild acute obstructive pancreatitis was present in two patients (2/4, 50%) with BGAs located in the ampulla region. During the follow-up period, one patient with a positive peripheral margin experienced tumor recurrence 2 years after the initial ESD. There was no disease-related death for the cohort. Conclusion: ESD was an effective and safe therapeutic option for BGA patients with excellent outcomes. Long-term follow-up is needed

Erratum: Atorvastatin Reduces Plaque Vulnerability in an Atherosclerotic Rabbit Model by Altering the 5-Lipoxygenase Pathway

<i>Objective:</i> The 5-lipoxygenase catalyzed formation of leukotriene lipid mediators is a mediator for inflammatory response in arteries. The present study investigated the relationship between atorvastatin and the 5-lipoxygenase pathway in an atherosclerotic rabbit model. <i>Methods:</i> Thirty male New Zealand White Rabbits were randomized into negative control, positive control and atorvastatin groups. At week 4, the rabbits were subjected to carotid balloon-dilation injury or carotid balloon-dilation injury, followed by treatment with atorvastatin. At week 12, all the animals were sacrificed. Plasma lipids, LTD₄, and 15-epi-lipoxin A₄ were measured using the enzymatic endpoint method and ELISA, respectively. RT-PCR was performed to detect the gene expression of 5-lipoxygenase-activating protein and cysLT1R in rabbit carotid arteries. Finally, histological analysis was used to evaluate the pathophysiological changes of rabbit carotid arteries. <i>Results:</i> The results showed atorvastatin markedly lowered serum lipids and LTD₄ levels compared with the control group. Similarly, mRNA expression of 5-lipoxygenase-activating protein and cysLT1R was significantly inhibited by atorvastatin. Decreased carotid plaque instability was evident in atorvastatin-treated animals, as demonstrated by a thickened elastic layer, less neointima hyperplasia and macrophage proliferation. <i>Conclusions:</i> Atorvastatin may stabilize carotid plaque by regulating the 5-lipoxygenase pathway in atherosclerotic rabbits and delay the progression of atherosclerosis by exerting anti-inflammatory effects

Supplementary Material for: General Anesthesia may have Similar Outcomes with Conscious Sedation in Thrombectomy Patients with Acute Ischemic Stroke: A Real-World Registry in China

<b><i>Background and Purpose:</i></b> Clinical trials showed that anesthesia may not influence the functional outcome in stroke patients with endovascular therapy; however, data are lacking in China. Using real-world registry data, our study aims to compare the effects of general anesthesia or conscious sedation on functional outcomes in stroke patients treated with thrombectomy in China. <b><i>Methods:</i></b> Consecutive patients with acute anterior circulation stroke receiving thrombectomy in 21 stroke centers between January 2014 and June 2016 were included in this study. The propensity score analysis with 1: 1 ratio was used to match the baseline variables between patients with general anesthesia and the conscious sedation. The 90-day modified Rankin Scale (mRS), symptomatic intracranial hemorrhage (sICH), and death were compared between groups. <b><i>Results:</i></b> Of the 698 patients undergoing endovascular treatment, 138 were treated with general anesthesia and 560 with conscious sedation. After propensity score matching, 114 general anesthesia and 114 conscious sedation patients were matched. The proportions of patients with 90-day mRS 0–2 were not significantly different between general anesthesia and conscious sedation groups (41.2% [47/114] vs. 46.5% [53/114], <i>p</i> = 0.470), nor were the rates of sICH (21.9% [25/114] vs. 12.3% [14/114], <i>p</i> = 0.072) and 90-day mortality (31.6% [36/114] vs. 21.9% [25/114], <i>p</i> = 0.145). <b><i>Conclusion:</i></b> Anesthesia patterns may have no significant impacts on clinical outcomes in patients with acute anterior circulation occlusion stroke undergoing endovascular treatment in the real-world practice in China

Supplementray Material for: Clinical Effectiveness and Safety Outcomes of Endovascular Treatment for Acute Anterior Circulation Ischemic Stroke in China

<strong><em>Backgrounds and Purpose:</em></strong> This study was aimed at investigating the outcomes and predictors for the poor functional outcome after endovascular treatment (EVT) in a large, mostly Asian population. <b><i>Methods:</i></b> Between January 2014 and June 2016, acute stroke patients with anterior circulation occlusion and EVT were retrospectively enrolled from 21 stroke centers in China. The main outcomes were modified Rankin Scale (0-2 as functional independence, 3-6 as poor) at 90 days, symptomatic intracranial hemorrhage (sICH) at 72 h, and death at 90 days. Logistic regression was used to identify predictors for poor functional outcome at 90 days. <b><i>Results:</i></b> Of the 698 patients, 304 (43.6%) patients had functional independence at 90 days. The sICH rate was 15.5% (108/698) and mortality rate at 90 days was 25.4% (177/698). Age (OR 1.04, 95% CI 1.02-1.07), National Institutes of Health Stroke Scale score at admission (11-20 vs. ≤10, OR 2.38, 95% CI 1.23-4.59; ≥21 vs. ≤10, OR 3.66, 95% CI 1.72-7.80), baseline<b> </b>glucose level (OR 1.09, 95% CI 1.01-1.18), onset to groin puncture >6 h (OR 1.88, 95% CI 1.06-3.31), sICH (OR 15.49, 95% CI 5.16-46.43), and pneumonia (OR 3.15, 95% CI 1.86-5.32) were independent predictors of poor functional outcomes, while good recanalization (OR 0.26, 95% CI 0.13-0.54), preoperative Alberta Stroke Program Early CT Score 8-10 (OR 0.48, 95% CI 0.28-0.83), and good collateral flow (OR 0.50, 95% CI 0.32-0.79) were protective factors. <b><i>Conclusions:</i></b> This study provides evidence in real world to support the performance of EVT in acute anterior circulation stroke patients in Chinese population. Patients with small infarct core, successful recanalization, good collateral status, and short treatment delay without sICH or pneumonia may benefit from EVT