Losartan alleviates renal fibrosis by inhibiting the biomechanical stress-induced epithelial-mesenchymal transition of renal epithelial cells.

Angiotensin receptor blockers (ARBs) have been reported to be beneficial of renal fibrosis, but the molecular and cellular mechanisms are still unclear. In this study, we investigated the effectiveness and relevant mechanism of ARBs in alleviating renal fibrosis, especially by focusing on biomechanical stress-induced epithelial to mesenchymal transition (EMT) of renal epithelial cells. Unilateral ureteral obstruction (UUO) renal fibrosis model was established in mice by ligating the left ureter, and then randomly received losartan at a low dose (1 mg/kg) or a regular dose (3 mg/kg) for 2 weeks. Compared to the control, histological analysis showed that losartan treatment at either a low dose or a regular dose effectively attenuated renal fibrosis in the UUO model. To further understand the mechanism, we ex vivo loaded primary human renal epithelial cells to 50 mmHg hydrostatic pressure. Western blot and immunostaining analyses indicated that the loading to 50 mmHg hydrostatic pressure for 24 h significantly upregulated vimentin, ß-catenin and α-SMA, but downregulated E-cadherin in renal epithelial cells, suggesting the EMT. The addition of 10 or 100 nM losartan in medium effectively attenuated the EMT of renal epithelial cells induced by 50 mmHg hydrostatic pressure loading. Our in vivo and ex vivo experimental data suggest that losartan treatment, even at a low dose can effectively alleviate renal fibrosis in mouse UUO model, at least partly by inhibiting the biomechanical stress-induced EMT of renal epithelial cells. A low dose of ARBs may repurpose for renal fibrosis treatment.

Real-time fluorescent cholangiography with indocyanine green in laparoscopic cholecystectomy: a randomized controlled trial to establish the optimal indocyanine green dose within 30 min preoperatively.

PURPOSE: To establish the optimal dose of indocyanine green (ICG) to administer intravenously 30 min before laparoscopic cholecystectomy (LC). METHODS: In this randomized controlled trial (RCT), patients undergoing LC for cholecystitis, cholelithiasis, and/or cholecystic polyps were randomized into four groups given four different ICG doses (0.025, 0.1, 0.25, 2.5 mg). Using OptoMedic endoscopy combined with a near-infrared fluorescent imaging system, we evaluated the fluorescence intensity (FI) of the common bile duct and liver at three timepoints: before surgical dissection of the cystohepatic triangle, before clipping of the cystic duct, and before closure. The bile duct-to-liver ratio (BLR) of the FI was analyzed to assess the cholangiography effect. RESULTS: Sixty-four patients were allocated to one of four groups, with 40 patients included in the final analysis. Generally, with increasing ICG doses, the levels of FI in the bile duct and liver increased gradually at each of the three timepoints. Before surgical dissection of the cystohepatic triangle, 0.1-mg ICG showed the highest BLR (F = 3.47, p = 0.0259). Before clipping the cystic duct and before closure, the 0.025- and 0.1-mg groups showed a higher BLR than the 0.25- and 2.5-mg groups (p < 0.05). When setting the ideal cholangiography at a BLR ≥ 1, ≥ 3, or ≥ 5, the 0.1-mg group showed the highest qualified case number at the three timepoints. CONCLUSIONS: The intravenous administration of 0.1-mg ICG, 30 min before LC, is significantly better for fluorescent cholangiography of the extrahepatic biliary structures before dissection and clipping of the cystohepatic triangle. TRIAL REGISTRATION: This study was registered in the Chinese Clinical Trial Registry (ChiCTR) (ChiCTR2200057933).

Angiotensin receptor blocker alleviates liver fibrosis by altering the mechanotransduction properties of hepatic stellate cells.

Angiotensin receptor blockers have been reported to be beneficial to liver fibrosis, but the relevant molecular and cellular mechanisms remain unclear. We herein investigated whether low-dose angiotensin receptor blocker alleviated liver fibrosis through mechanotransduction regulation. Hydrostatic pressure-induced liver fibrosis model was established in mice by ligating partially the inferior vena cava, and then randomly received a very low dose of losartan (0.5 mg/kg) or placebo treatment for 8 weeks. We found that losartan administration interfered the expression of several mechanotransductive molecules, and effectively alleviated liver fibrosis. Using a commercial device, we further confirmed that ex vivo loading of hepatic stellate cells to 50 mmHg hydrostatic pressure for 24 h significantly upregulated RhoA, ROCK, AT1R, and p-MLC2, which was effectively attenuated by adding 10 nM losartan in medium. Our in vivo and ex vivo experimental data suggest that low-dose angiotensin receptor blockers may alleviate hydrostatic pressure-induced liver fibrosis by altering the mechanotransduction properties of hepatic stellate cells.NEW & NOTEWORTHY Our ex vivo and in vivo experiments clearly indicated that low-dose losartan alleviated liver fibrosis, likely by modulating the mechanotransduction properties of HSCs. Uncovering the biomechanical signaling pathway of ARB treatment on liver fibrosis will be helpful to develop novel molecular targeting therapy for liver diseases.

Bioengineering of a CLiP-derived tubular biliary-duct-like structure for bile transport in vitro.

The integration of a bile drainage structure into engineered liver tissues is an important issue in the advancement of liver regenerative medicine. Primary biliary cells, which play a vital role in bile metabolite accumulation, are challenging to obtain in vitro because of their low density in the liver. In contrast, large amounts of purified hepatocytes can be easily acquired from rodents. The in vitro chemically induced liver progenitors (CLiPs) from primary mature hepatocytes offer a platform to produce biliary cells abundantly. Here, we generated a functional CLiP-derived tubular bile duct-like structure using the chemical conversion technology. We obtained an integrated tubule-hepatocyte tissue via the direct coculture of hepatocytes on the established tubular biliary-duct-like structure. This integrated tubule-hepatocyte tissue was able to transport the bile, as quantified by the cholyl-lysyl-fluorescein assay, which was not observed in the un-cocultured structure or in the biliary cell monolayer. Furthermore, this in vitro integrated tubule-hepatocyte tissue exhibited an upregulation of hepatic marker genes. Together, these findings demonstrated the efficiency of the CLiP-derived tubular biliary-duct-like structures regarding the accumulation and transport of bile.

Chemical conversion of aged hepatocytes into bipotent liver progenitor cells.

AIM: In the aging society, understanding the influence of hepatocyte age on hepatocyte donation may inform efforts to expand alternative cell sources to mitigate liver donor shortage. A combination of the molecules Y27632, A-83-01, and CHIR99021 has been used to reprogram rodent young hepatocytes into chemically induced liver progenitor (CLiP) cells; however, whether it could also reprogram aged hepatocytes has not yet been elucidated. METHODS: Primary hepatocytes were isolated from aged and young donor rats, respectively. Hepatic histological changes were evaluated. Differences in gene expression in hepatocytes were identified. The in vitro reprogramming plasticity of hepatocytes as evidenced by CLiP conversion and the hepatocyte and cholangiocyte maturation capacity of reprogrammed CLIPs were analyzed. The effect of hepatocyte growth factor (HGF) on cell propagation was also investigated. RESULTS: The histological findings revealed ongoing liver damage with inflammation, fibrosis, senescence, and ductular reaction in aged livers. Microarray analysis showed altered gene expression profiles in hepatocytes from aged donors, especially with regard to metabolic pathways. Aged hepatocytes could be converted into CLiPs (Aged-CLiPs) expressing progenitor cell markers, but with a relatively low proliferative rate compared with young hepatocytes. Aged-CLiPs possessed both hepatocyte and cholangiocyte maturation capacity. HGF facilitated CLiP conversion in aged hepatocytes, which was partly related to the activation of Erk1 and Akt1 signaling. CONCLUSIONS: Aged rat hepatocytes have retained reprogramming plasticity as evidenced by CLiP conversion in culture. HGF promoted proliferation and CLiP conversion in aged hepatocytes. Hepatocytes from aged donors may be used as an alternative cell source to mitigate donor shortage.

Extrahepatic biliary tract visualization using near-infrared fluorescence imaging with indocyanine green: optimization of dose and dosing time.

BACKGROUND: The dose and dosing time of indocyanine green (ICG) vary among fluorescence cholangiography (FC) studies. The purpose of this prospective, randomized, exploratory clinical trial was to optimize the dose and dosing time of ICG. METHODS: PubMed was searched to determine the optimal dose. To optimize the dosing time of ICG, a clinical trial was designed with two parts. The first part included patients with T tubes for more than 1 month. After the patient was injected with ICG, bile was collected at 10 time points to explore the change and trends of bile fluorescence intensity (FI). In addition, the results of the first experiment were used to setup a randomized controlled trial (RCT) that aimed to find the optimal dosing timing for ICG injections for laparoscopic cholecystectomy (LC). During surgery, imaging data were collected for analysis. RESULTS: After performing a systematic review, the ICG injection dose for each patient in the clinical trial was 10 mg. Five patients were included in the first part of the study. Bile collected 8 h after ICG injection had a higher FI than bile collected at other time points (p < 0.05), and the FI of bile collected 20 h after ICG injection was nearly zero. In the second part of the experiment, 4 groups of patients (6 patients per group) were injected with 10 mg ICG at 8, 10, 12 and 14 h prior to surgery. The distribution of bile duct FI (p = 0.001), liver FI (p < 0.001), and common bile duct (CBD)-to-liver contrast (p = 0.001) were not the same in each group. Further analysis with the Bonferroni method revealed the following: (1) the FI of the CBD in the 8 h group was significantly different from that in the 14 h group (adjusted p < 0.001); (2) the liver FI of the 8 h group was higher than that of the 10 h group (adjusted p = 0.042) and the 14 h group (adjusted p < 0.001); and (3) the CBD-to-liver contrast of the 8 h group was lower than that of the 10 h group (adjusted p = 0.013) and the 14 h group (adjusted p = 0.001). CONCLUSION: ICG FC enables the real-time identification of extrahepatic bile ducts. The optimal effect of FC can be achieved by performing 10 mg ICG injections 10 to 12 h prior to surgery.

3D hESC exosomes enriched with miR-6766-3p ameliorates liver fibrosis by attenuating activated stellate cells through targeting the TGFßRII-SMADS pathway.

BACKGROUND: Exosomes secreted from stem cells exerted salutary effects on the fibrotic liver. Herein, the roles of exosomes derived from human embryonic stem cell (hESC) in anti-fibrosis were extensively investigated. Compared with two-dimensional (2D) culture, the clinical and biological relevance of three-dimensional (3D) cell spheroids were greater because of their higher regeneration potential since they behave more like cells in vivo. In our study, exosomes derived from 3D human embryonic stem cells (hESC) spheroids and the monolayer (2D) hESCs were collected and compared the therapeutic potential for fibrotic liver in vitro and in vivo. RESULTS: In vitro, PKH26 labeled-hESC-Exosomes were shown to be internalized and integrated into TGFß-activated-LX2 cells, and reduced the expression of profibrogenic markers, thereby regulating cellular phenotypes. TPEF imaging indicated that PKH26-labeled-3D-hESC-Exsomes possessed an enhanced capacity to accumulate in the livers and exhibited more dramatic therapeutic potential in the injured livers of fibrosis mouse model. 3D-hESC-Exosomes decreased profibrogenic markers and liver injury markers, and improved the level of liver functioning proteins, eventually restoring liver function of fibrosis mice. miRNA array revealed a significant enrichment of miR-6766-3p in 3D-hESC-Exosomes, moreover, bioinformatics and dual luciferase reporter assay identified and confirmed the TGFßRII gene as the target of miR-6766-3p. Furthermore, the delivery of miR-6766-3p into activated-LX2 cells decreased cell proliferation, chemotaxis and profibrotic effects, and further investigation demonstrated that the expression of target gene TGFßRII and its downstream SMADs proteins, especially phosphorylated protein p-SMAD2/3 was also notably down-regulated by miR-6766-3p. These findings unveiled that miR-6766-3p in 3D-hESC-Exosomes inactivated SMADs signaling by inhibiting TGFßRII expression, consequently attenuating stellate cell activation and suppressing liver fibrosis. CONCLUSIONS: Our results showed that miR-6766-3p in the 3D-hESC-Exosomes inactivates smads signaling by restraining TGFßRII expression, attenuated LX2 cell activation and suppressed liver fibrosis, suggesting that 3D-hESC-Exosome enriched-miR-6766-3p is a novel anti-fibrotic therapeutics for treating chronic liver disease. These results also proposed a significant strategy that 3D-Exo could be used as natural nanoparticles to rescue liver injury via delivering antifibrotic miR-6766-3p.

Relation of AURKB over-expression to low survival rate in BCRA and reversine-modulated aurora B kinase in breast cancer cell lines.

BACKGROUND: New therapeutic drug for breast cancer (BRCA), especially triple negative BRCA (TNBC), is urgently needed. Even though 2-(4-morpholinoanilino)-6-cyclohexylaminopurine (reversine) is an aurora kinase inhibitor, it also inhibits some cancer cells and human BRCA cells. However, the potential roles of reversine as a novel therapeutic agent for the treatment of BRCA remains unknown and must be further investigation. Thus, the relationship of reversine to aurora kinase in BCRA has not been reported. The relationship between AURKB and survival rate in BRCA has never been reported. Herein, we tested the roles of reversine on different BRCA cell line subtypes. We also investigated the relationship between AURKB and survival rate in BRCA as well as reversine to Aurora kinase expression in BCRA cell lines, including TNBC subtype, 4T1, MDA-MB-231, and luminal subtype MCF-7. METHODS: Cell viability and apoptosis were detected using Cell Counting Kit-8 and flow cytometry analysis, respectively. Apoptotic and tumor-related proteins were tested using Western blot analysis. Important microRNAs that regulate BRCA were analyzed using RT-PCR. UALCAN public databases were used to analyze the targeted gene profiles, and the PROGgeneV2 database was used to study the prognostic implications of genes. RESULTS: Reversine inhibits cell proliferation and induces cell apoptosis by modulating caspase-3 and bax/bcl-2 among the three cell lines. Data from the UALCAN public database show that BRCA tissues expressed high gene levels of AURKB, TIMP1, MMP9, and TGFB1 compared with the normal tissue. Among the over-expressed genes in BRCA, AURKB ranks 9th in TNBC, 49th in luminal subtype, and 48th in HER2 subtype. High AURKB level in BRCA is highly related to the low survival rate in patients displayed in 18 databases searched via PROGgeneV2. The protein levels of aurora B kinase (Aurora B), which is encoded by AURKB gene, are highly suppressed by reversine in the three cell lines. The tumor-related proteins TGF-ß1, TIMP1, and MMP9 are partially suppressed by reversine but with different sensitivity in the three cell lines. The reversine-affected microRNAs, such as miR129-5p, miR-199a-3p, and miR-3960, in MDA-MB-231 cell line might be the research targets in TNBC regulation. CONCLUSIONS: In BRCA, the level of AURKB are over-expressed and is related to low survival rate. Reversine contributes to anti-growth effect in BRCA cell lines, especially for TNBC, by modulating the aurora B. However, the invasiveness, metastasis, and anti-tumor effects of reversine in vivo and in vitro must be further investigated.

Effect of reversine on cell cycle, apoptosis, and activation of hepatic stellate cells.

Experimental and clinical evidence show that liver fibrosis is potentially reversible. Hepatic stellate cells (HSCs) play a key role in the development of liver fibrosis. Some studies have shown that reversine could induce cell apoptosis. We attempted to elucidate the effect of reversine on cell cycle, apoptosis, and activation of HSCs. Data showed that reversine induced morphological changes in HSCs, inhibited cell proliferation, and induced cell-cycle arrest at the G2/M phase. Reversine induced cell apoptosis through caspase-dependent and mitochondria-dependent pathways. Reversine inhibited the activation of HSCs through TGF-ß signaling pathway and degraded extracellular matrix protein collagen-I. The decreased TIMP1 and TGF-ß1 proteins promoted fibrosis reversion. Reversine might be a promising drug for liver fibrosis reversion because it induces HSCs apoptosis, restrains cell proliferation, reduces HSCs activation, and degrades extracellular matrix in vitro.

Short-term administration of steroids does not affect postoperative complications following liver resection: Evidence from a meta-analysis of randomized controlled trials.

AIM: Although perioperative short-term administration of steroids can attenuate surgical stress response following liver resection, there is no consensus concerning the effect on postoperative complications. This study aims to use meta-analysis to quantitatively investigate the effect of perioperative short-term administration of steroids on postoperative complications following liver resection. METHODS: A systematic published work search was performed to detect randomized controlled trials (RCT) assessing the effect of perioperative short-term administration of steroids on outcomes following liver resection. Parameters of surgical stress, hospital stay and postoperative complications were analyzed. Two authors independently assessed study quality and extracted data. All data were analyzed using RevMan version 5 and meta-analyses were performed using a random-effects model. RESULTS: Five RCT published between 2001 and 2011 containing a total of 379 patients were eligible for final analysis. Serum total bilirubin, interleukin-6 and C-reactive protein were significantly lower in the steroid than in the control group on postoperative day 1 (P = 0.02, 0.004 and 0.02, respectively). There was no difference in duration of hospital stay between the steroid and control group (P = 0.37). The analysis of end-points including infective complications (odds ratio [OR], 0.95), wound complications (OR, 0.67), bile leakage (OR, 0.58) and overall complications (OR, 0.50) revealed no difference between steroid administration and no treatment. There was no postoperative death or adverse effect attributable to steroid treatment in all patients. CONCLUSION: On currently available evidence, short-term administration of steroids does not increase incidence of complications in patients undergoing liver resection.

Metabolic signatures of lymphangioleiomyomatosis in biofluids: nuclear magnetic resonance (NMR)-based metabonomics of blood plasma: a case-control study.

Background: Our aim was to analyze and compare the characteristics and differences of blood metabolites between lymphangioleiomyomatosis (LAM) patients and healthy controls, in order to find biomarkers that can be used for the diagnosis and classification of LAM. Methods: Between January 2020 to January 2022, 61 eligible LAM patients [51 sporadic LAM (S-LAM) and 10 tuberous sclerosis complex LAM (TSC-LAM)] from the First Affiliated Hospital of Guangzhou Medical University and 30 healthy controls were enrolled. Blood samples were taken for nuclear magnetic resonance (NMR) detection. Data analysis was performed by the umbrella program, and Wilcoxon analysis was used for comparisons between groups. The difference indicators were modeled by logistic regression. Diagnostic accuracy of the best predictive parameters was evaluated by the area under the receiver operating characteristic (ROC) curve (AUC), and the sensitivity and specificity were calculated. Results: The indexes differed between LAM patients and healthy controls, S-LAM patients and healthy controls, and between TSC-LAM patients and healthy controls. There were two different metabolic indexes between S-LAM and TSC-LAM patients. After logistic regression modeling and ROC analysis, methionine (AUC =0.929, sensitivity =73.8%, specificity =100%, cut-off value =0.011 mmol/L) and acetic acid (AUC =0.966, sensitivity =95.1%, specificity =90%, cut-off value =0.006 mmol/L) had the highest diagnostic efficiency in LAM patients, and could be used to distinguish between affected and healthy people. Methionine was significantly associated with pneumothorax (P<0.05), and creatinine was significantly correlated with hysteromyoma (P<0.05). Conclusions: Methionine and acetic acid in the plasma of LAM patients are potential biomarkers. Methionine was also associated with pneumothorax in LAM patients. Also, acetone and creatinine were promising metabolic markers to distinguish S-LAM from TSC-LAM. NMR as a new non-invasive diagnostic method had a good discriminatory power for LAM.

Predictive value of ELWI combined with sRAGE/esRAGE levels in the prognosis of critically ill patients with acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a life-threatening condition. Accurate judgement of the disease progression is essential for controlling the condition in ARDS patients. We investigated whether changes in the level of serum sRAGE/esRAGE could predict the 28-day mortality of ICU patients with ARDS. A total of 83 ARDS patients in the ICU of the Second Affiliated Hospital of Nantong University from January 2021 to June 2022 were consecutively enrolled in this study. Demographic data, primary diagnosis and comorbidities were obtained. Multiple scoring systems, real-time monitoring systems, and biological indicators were determined within 6 h of admission. The clinical parameters for survival status of the ARDS patients were identified by multivariate logistic regression. Receiver operating characteristic (ROC) curve analysis was employed to verify the accuracy of the prognosis of the related parameters. The admission level of sRAGE was significantly higher in the nonsurvival group than in the survival group (p < 0.05), whereas the serum esRAGE level showed the opposite trend. Multivariate logistic regression analysis showed that sRAGE (AUC 0.673, p < 0.05), esRAGE (AUC 0.704, p < 0.05), and ELWI (extravascular lung water index) (AUC 0.717, p < 0.05) were independent risk factors for the prognosis of ARDS. Model B (ELWI + esRAGE) could not be built as a valid linear regression model (ELWI, p = 0.079 > 0.05). Model C (esRAGE + sRAGE) was proven to have no significance because it had a predictive value similar to that of the serum levels of esRAGE (Z = 0.993, p = 0.351) or sRAGE (Z = 1.116, p = 0.265) alone. Subsequently, Model D (sRAGE + esRAGE + ELWI) showed the best 28-day mortality predictive value with a cut-off value of 0.426 (AUC 0.841; p < 0.001), and Model A (sRAGE + ELWI) had a cut-off value of 0.401 (AUC 0.820; p < 0.001), followed by sRAGE (AUC 0.704, p = 0.004), esRAGE (AUC 0.717, p = 0.002), and ELWI (AUC 0.637, p = 0.028). In addition, there was no statistically significant difference between Model A and Model D (Z = 0.966, p = 0.334). The admission level of sRAGE was higher in the nonsurvival group, while the serum esRAGE level showed the opposite trend. Model A and Model D could be used as reliable combined prediction models for predicting the 28-day mortality of ARDS patients.

Reversine attenuates cholestatic ductular reaction in rats.

Ductular reaction (DR) is usually observed in biliary disorders or various liver disorders, including nonalcoholic fatty liver disease. Few studies have focused on interrupting the DR process in the cholestatic environment. Here, we investigated the impact of reversine on DR in rats that had undergone bile duct ligation (BDL). Cholestatic injury was induced in rats 2 weeks following BDL. DR was assessed with biliary markers by immunohistochemistry. Biliary epithelial cells (BECs) were isolated for the analysis of proliferation and biliary factor gene expression. The effects of reversine on DR and fibrosis were analyzed in vivo via intraperitoneal injection in rats for 2 weeks. Chemically-induced BEC formation was used to investigate the biliary markers affected by reversine in vitro. DR with increased BEC expansion was identified in cholestatic liver injury, as indicated by CK7, CK19, and EpCAM expression around the portal vein in BDL rats. BDL-induced DR cells showed the increased expression of genes regulating cell proliferation (Ki67, Foxm1, and Pcna) and biliary markers (Krt7, Krt19, Epcam, Sox9, Cftr, and Asbt). Reversine attenuated cholestatic fibrosis and DR in rats. Reversine affected chemically-induced BEC formation, with the decreased expression of biliary Krt7, Cftr, and Ggt1 genes in vitro. BDL-induced Notch activation was attenuated upon reversine treatment in vivo, in part via the Notch/Sox9 pathway. In conclusion, reversine attenuated cholestatic ductular reaction and fibrosis in rats and reduced the bile duct formation associated with Dlk1/Notch/Sox9 signaling. Reversine may be regarded as a potential drug for cholangiopathies for preventing a ductular reaction.

Community structure characteristics of ichthyoplankton during the water-sediment regulation scheme in the Yellow River in 2020 and 2021.

The Water-Sediment Regulation Scheme (WSRS) will deliver large amounts of water and sand to the Yellow River basin within a short period of time. This will significantly change the physicochemical environment of the Yellow River estuary and the surrounding marine ecosystem. Its effects on the spatial and temporal distribution patterns of ichthyoplankton are still unknown. In this study, six surface horizontal trawl surveys of ichthyoplankton were conducted during the WSRS in 2020 and 2021 using plankton nets. The results were as follows: (1) the estuarine sedentary fish Cynoglossus joyeri was the main species controlling the succession pattern of summer ichthyoplankton communities in the Yellow River estuary. (2) The WSRS influenced the ichthyoplankton community structure by changing the runoff, salinity, and suspension environment in the estuary. (3) The northern and southeastern parts of the estuary near Laizhou Bay were the main aggregation areas of the ichthyoplankton community.

Three-dimensional human bile duct formation from chemically induced human liver progenitor cells.

Background: The intrahepatic bile ducts (BDs) play an important role in the modification and transport of bile, and the integration between the BD and hepatocytes is the basis of the liver function. However, the lack of a source of cholangiocytes limits in vitro research. The aim of the present study was to establish three-dimensional BDs combined with human mature hepatocytes (hMHs) in vitro using chemically induced human liver progenitor cells (hCLiPs) derived from hMHs. Methods: In this study, we formed functional BDs from hCLiPs using hepatocyte growth factor and extracellular matrix. BDs expressed the typical biliary markers CK-7, GGT1, CFTR and EpCAM and were able to transport the bile-like substance rhodamine 123 into the lumen. The established three-dimensional BDs were cocultured with hMHs. These cells were able to bind to the BDs, and the bile acid analog CLF was transported from the culture medium through the hMHs and accumulated in the lumen of the BDs. The BDs generated from the hCLiPs showed a BD function and a physiological system (e.g., the transport of bile within the liver) when they were connected to the hMHs. Conclusion: We present a novel in vitro three-dimensional BD combined with hMHs for study, drug screening and the therapeutic modulation of the cholangiocyte function.

Establishment of a prognostic model based on m6A regulatory factors and stemness of hepatocellular carcinoma using RNA-seq data and scRNA-seq data.

BACKGROUND: Hepatocellular carcinoma (HCC) with high incidence and mortality is one of the most common malignant cancers worldwide. Increasing evidence has reported that N6-methyladenosine (m6A) modification has been considered as a major contribution to the occurrence and development of tumors. METHOD: In our study, we comprehensively analyzed the connection between m6A regulatory factors and cancer stem cells (CSCs) of HCC to establish a clinical tool for predicting its outcome. First, we concluded that the expression level of m6A regulatory factors was related with the stemness of hepatocellular carcinoma. Subsequently, we gained a ten hub regulatory factors that were associated with prognosis of hepatocellular carcinoma by overall survival (OS) analysis using ICGC and TCGA datasets, and these regulatory factors included YTHDF1, IGF2BP1, METTL3, IGF2BP3, HNRNPA2B1, IGF2BP2, RBM15B, HNRNPC, RBMX, and LRPPR. Next, we found that these ten hub m6A regulatory factors were highly expressed in CSCs, and CSCs related pathways were also enriched by the gene set variation analysis (GSVA). Then, correlation, consensus clustering and PCA analysis were performed to reveal potential therapeutic benefits of HCC. Moreover, univariate Cox regression (UNICOX), LASSON and multivariate Cox regression (MULTICOX) analyses were adopted to establish HCC prognosis prediction signature. RESULTS: Four regulatory factors RBM15B, LRPPRC, IGF2BP1, and IGF2BP3 were picked as valuable prognostic indicators. CONCLUSION: In summary, these ten hub regulatory factors would be useful therapeutic targets for HCC treatment, and RBM15B/LRPPRC/IGF2BP1/IGF2BP3 prognostic indicators can be used to guide therapy for HCC patients.

Correlation analysis between plasma fibrinogen and nerve electrophysiological changes in type 2 diabetic peripheral neuropathy.

INTRODUCTION: The aim of the study was to investigate the pathogenesis of diabetic peripheral neuropathy (DPN) and the value of fibrinogen (FIB) in the early diagnosis of DPN. MATERIAL AND METHODS: A total of 121 patients with type 2 diabetes mellitus (T2DM) and DPN hospitalized in the Endocrinology Department of the 923 Hospital of the People's Liberation Army of China were randomly selected between May and October 2020 and divided into a T2DM asymptomatic (no peripheral neuropathy-related symptoms) group (66 cases) and a T2DM symptomatic group (55 cases) according to the presence or absence of clinical neurological symptoms and signs. Forty healthy volunteers were selected as a normal control group. In addition to plasma FIB and nerve electrophysiological tests, all included subjects were electrophysiologically tested for nerve conduction velocity (NCV), terminal motor latency (DML), sensory nerve action potential (SNAP) amplitude, and compound muscle action potential (CMAP) amplitude. RESULTS: Compared with the control group, NCV was slowed down in T2DM patients, DML was prolonged, and the amplitude of CMAP and SNAP were decreased. Compared with asymptomatic T2DM patients, symptomatic patients had slower NCV, longer DML, lower CMAP amplitude of median nerve, ulnar nerve and tibial nerve, and significantly lower SNAP amplitude of median nerve and ulnar nerve. CMAP amplitudes were decreased, and median and ulnar nerve SNAP amplitudes were also significantly decreased ( p < 0.05). The plasma FIB concentration of asymptomatic patients with T2DM was higher than that of the control group, and the plasma FIB concentration of symptomatic patients with T2DM was higher than that of asymptomatic patients with T2DM ( p < 0.01). The NCV and DML of asymptomatic patients with T2DM slowed down and prolonged as the FIB level increased; the NCV of T2DM symptomatic patients also slowed down as FIB increased, and median and ulnar nerve DML increased as FIB increased. There was no correlation between NCV and DML and the plasma FIB level in the control group. SNAP amplitudes of symptomatic and asymptomatic patients with T2DM decreased as plasma FIB increased, while CMAP amplitudes of the tibial nerve and the T2DM symptomatic ulnar nerve decreased as FIB increased in the control group. CONCLUSIONS: FIB may be a contributing factor for diabetic neuropathy and could be used as an indicator in the early screening and diagnosis of peripheral neuropathy in patients with T2DM.

Investigation of the optimal indocyanine green dose in real-time fluorescent cholangiography during laparoscopic cholecystectomy with an ultra-high-definition 4K fluorescent system: a randomized controlled trial.

This study aimed to investigate the indocyanine green (ICG) dose in real-time fluorescent cholangiography during laparoscopic cholecystectomy (LC) with a 4K fluorescent system. A randomized controlled clinical trial was conducted in patients who underwent LC for treatment of cholelithiasis. Using the OptoMedic 4K fluorescent endoscopic system, we compared four different doses of ICG (1, 10, 25, and 100 µg) administered intravenously within 30 min preoperatively and evaluated the fluorescence intensity (FI) of the common bile duct and liver background and the bile-to-liver ratio (BLR) of the FI at three timepoints: before surgical dissection of the cystohepatic triangle, before clipping the cystic duct, and before closure. Forty patients were randomized into four groups, and 33 patients were fully analyzed, with 10 patients in Group A (1 µg), 7 patients in Group B (10 µg), 9 patients in Group C (25 µg), and 7 patients in Group D (100 µg). The preoperative baseline characteristics were compared among groups (p > 0.05). Group A showed no or minimal FI in the bile duct and liver background, while Group D showed extremely high FIs in the bile duct and in the liver background at the three timepoints. Groups B and C presented with visible FI in the bile duct and low FI in the liver background. With increasing ICG doses, the FIs in the liver background and bile duct gradually increased at the three timepoints. The BLR, however, showed no increasing trend with an increasing ICG dose. A relatively high BLR on average was found in Group B, without a significant difference compared to the other groups (p > 0.05). An ICG dose ranging from 10 to 25 µg by intravenous administration within 30 min preoperatively was appropriate for real-time fluorescent cholangiography in LC with a 4K fluorescent system. Registration: This study was registered in the Chinese Clinical Trial Registry (ChiCTR No: ChiCTR2200064726).