Role of Fibrinogen in Type-2 Diabetes Mellitus with Diabetic Neuropathy and its Preliminary Mechanism.

INTRODUCTION: Diabetic peripheral neuropathy (DN) is the most common complication of type 2 diabetes mellitus (T2DM). OBJECTIVE: This study aimed to explore the role of fibrinogen (FIB) in T2DM neuropathy and its preliminary mechanism. METHODS: Ten male Sprague-Dawley rats were divided into a normal control group (NC group) and a T2DM neuropathy model group (DN group). The DN group was given a high-energy diet and streptozotocin, while the NC group was given a normal diet and a citric acid buffer. The expression levels of related proteins were analysed. RESULTS: Electrophysiology: Compared with the NC group, the conduction latency of the somatosensory-evoked potential and nerve conduction velocity was prolonged in the DN group, while the motor nerve action potential was decreased. As seen under a light microscope, the peripheral nerve fibres in the DN group were swollen, and the nerve fibres in the posterior funiculus of the spinal cord were loose or missing. Moreover, as seen under an electron microscope, the peripheral nerve demyelination of the DN group was severe, with microvascular blood coagulation, luminal stenosis, and collapse. Compared with the NC group, in the DN group, the expression of FIB was positively correlated with the expression of both ionised calcium-binding adaptor molecule-1 and glial fibrillary acidic protein. Compared with the NC group, in the DN group, the expression of platelet/endothelial cell adhesion molecule-1 and B-cell lymphoma 2 was negatively correlated. CONCLUSION: The increased concentration of FIB may be the cause of neuropathy, and its mechanism may be related to its promotion of inflammatory response, blood coagulation, and vascular stenosis.

Recent discoveries in microbiota dysbiosis, cholangiocytic factors, and models for studying the pathogenesis of primary sclerosing cholangitis.

Primary sclerosing cholangitis (PSC) is a cholangiopathy caused by genetic and microenvironmental changes, such as bile homeostasis disorders and microbiota dysbiosis. Therapeutic options are limited, and proven surveillance strategies are currently lacking. Clinically, PSC presents as alternating strictures and dilatations of biliary ducts, resulting in the typical "beaded" appearance seen on cholangiography. The pathogenesis of PSC is still unclear, but cholangiocytes play an essential role in disease development, wherein a reactive phenotype is caused by the secretion of neuroendocrine factors. The liver-gut axis is implicated in the pathogenesis of PSC owing to the dysbiosis of microbiota, but the underlying mechanism is still poorly understood. Alterations in cholangiocyte responses and related signalling pathways during PSC progression were elucidated by recent research, providing novel therapeutic targets. In this review, we summarise the currently known underlying mechanisms of PSC pathogenesis caused by the dysbiosis of microbiota and newly reported information regarding cholangiocytes in PSC. We also summarise recently reported in vitro and in vivo models for studying the pathogenesis of PSC.

FoxO3 restricts liver regeneration by suppressing the proliferation of hepatocytes.

Upon injury, the liver is capable of substantial regeneration from the original tissue until an appropriate functional size. The underlying mechanisms controlling the liver regeneration processes are not well elucidated. Previous studies have proposed that the transcription factor FoxO3 is involved in various liver diseases, but its exact role in the regulation of liver regeneration remains largely unclear. To directly test the detailed role of FoxO3 in liver regeneration, both a constitutive Albumin-Cre driver line and adeno-associated virus serotype 8 (AAV8)-Tbg-Cre (AAV-Cre)-injected adult FoxO3fl/fl mice were subjected to 70% partial hepatectomy (PH). Our data demonstrate that FoxO3 deletion accelerates liver regeneration primarily by limiting polyploidization and promoting the proliferation of hepatocytes during liver regeneration. RNA-seq analysis indicates that FoxO3 deficiency greatly alters the expression of gene sets associated with cell proliferation and apoptosis during liver regeneration. Chromatin immunoprecipitation-PCR (ChIP-PCR) and luciferase reporter assays reveal that FoxO3 promotes the expression of Nox4 but suppresses the expression of Nr4a1 in hepatocytes. AAV8 virus-mediated overexpression of Nox4 and knockdown of Nr4a1 significantly suppressed hepatocyte proliferation and liver regeneration in FoxO3-deficient mice. We demonstrate that FoxO3 negatively controls hepatocyte proliferation through Nox4 upregulation and Nr4a1 downregulation, thereby ensuring appropriate functional regeneration of the liver. Our findings provide novel mechanistic insight into the therapeutic mechanisms of FoxO3 in liver damage and repair.

Laminin alpha-3 and thrombospondin-1 differently regulate the survival and differentiation of hepatocytes and hepatic stem cells from neonatal mice.

The aim of this study was to search and identify the extracellular matrix/adhesion molecules potentially regulating liver regeneration. By using pathway-focused PCR array, we investigated the dynamic changes in the expression of extracellular matrix and adhesion molecules in normal livers or cholestatic livers following partial hepatectomy in adult mice. To confirm the data from PCR array, we further evaluated how laminin alpha-3 and thrombospondin-1 mediate the survival and differentiation of matured hepatocytes and immature hepatic stem cells by using primarily isolated liver cells from neonatal mice. According to the different changes in the expression of extracellular matrix and adhesion molecules between normal livers and cholestatic livers, we could find a number of potential molecules involved in liver regeneration. Our in vitro evaluations indicated that laminin alpha-3 significantly increased the number of liver cells (P<0.01 vs. Control) but decreased the proportion of claudin-3-positive hepatic stem cells (P<0.05 vs. Control). In contrast, thrombospondin-1 significantly reduced cell apoptosis (P<0.05 vs. Control) and maintained the proportion of claudin-3-positive hepatic stem cells. Otherwise, the combination of laminin alpha-3 and thrombospondin-1 increased the proliferation of liver cells. Based on our data, laminin alpha-3 and trombospondin-1 differently regulate the survival and differentiation of hepatocytes and hepatic stem cells, but relevant mechanisms are required to be elucidated by further study.

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.

Regenerative medicine for the hepatobiliary system: A review.

Liver transplantation, the only proven treatment for end-stage liver disease and acute liver failure, is hampered by the scarcity of donors. Regenerative medicine provides an alternative therapeutic approach. Tremendous efforts dedicated to liver regenerative medicine include the delivery of transplantable cells, microtissues, and bioengineered whole livers via tissue engineering and the maintenance of partial liver function via extracorporeal support. This brief review summarizes the current status of regenerative medicine for the hepatobiliary system. For liver regenerative medicine, the focus is on strategies for expansion of transplantable hepatocytes, generation of hepatocyte-like cells, and therapeutic potential of engineered tissues in liver disease models. For biliary regenerative medicine, the discussion concentrates on the methods for generation of cholangiocyte-like cells and strategies in the treatment of biliary disease. Significant advances have been made in large-scale and long-term expansion of liver cells. The development of tissue engineering and stem cell induction technology holds great promise for the future treatment of hepatobiliary diseases. The application of regenerative medicine in liver still lacks extensive animal experiments. Therefore, a large number of preclinical studies are necessary to provide sufficient evidence for their therapeutic effectiveness. Much remains to be done for the treatment of hepatobiliary diseases with regenerative medicine.

Differentiation of chemically induced liver progenitor cells to cholangiocytes: Investigation of the optimal conditions.

Chemically induced liver progenitor (CLiP) cells, converted in vitro from mature hepatocytes, possess the bipotentiality to differentiate into both hepatocytes and cholangiocytes. Here, we aimed to investigate the optimal conditions for bile duct (BD) induction from rat CLiPs. A two-step induction protocol was used for the differentiation of cholangiocytes. We investigated the effects of passage number, preincubation times, Matrigel, and mouse embryonic fibroblast (MEF) feeder cells on the induction of cholangiocytes. Earlier passages of CLiPs were better for BD induction compared with stable CLiPs. Extending the preincubation time of CLiPs before induction delayed the formation of the BD. Matrigel provided cells with space to form three-dimensional (3D) structures, but the long-term use of Matrigel from the induction step did not benefit the differentiation of CLiPs to cholangiocytes. MEF feeder cells, through the Jag/Notch pathway, affected BD formation and function, as well as gene and protein expression. CLiPs were a good cell source for cholangiocyte differentiation under appropriate conditions and may offer a key vehicle for the study of cholangiopathies in vitro.

Reversine and herbal Xiang-Sha-Liu-Jun-Zi decoction ameliorate thioacetamide-induced hepatic injury by regulating the RelA/NF-κB/caspase signaling pathway.

This study investigated the anti-fibrotic effects of reversine and Chinese medicine Xiang-Sha-Liu-Jun-Zi decoction (XSLJZD) on thioacetamide (TAA)-induced hepatic injury. Sprague-Dawley rats were intraperitoneally administered with TAA, then injected with reversine intraperitoneally, and/or orally provided with XSLJZD. TAA resulted in liver injury with increases in the liver index and levels of serum aspartate aminotransferase (AST) and alanine aminotransferase. Reversine alleviated the liver index and AST level and improved TAA-induced pathological changes but decreased TAA-induced collagen deposition, and α-smooth muscle actin and transforming growth factor-ß1 expression. Reversine also modulated the mRNA levels of inflammatory cytokines, such as RelA, interleukin (IL)-17A, IL-22, IL-1ß, IL-6, NLR family pyrin domain containing 3, platelet-derived growth factor, and monocyte chemoattractant protein, and suppressed nuclear factor (NF)-κB (p65) phosphorylation and caspase 1 activation. Meanwhile, XSLJZD protected TAA-injured liver without increasing fibrosis and enhanced the regulating effect of reversine on RelA, IL-17A, IL-1ß, and MCP-1 cytokines. In conclusion, reversine ameliorates liver injury and inhibits inflammation reaction by regulating NF-κB, and XSLJZD protects the liver through its synergistic effect with reversine on regulating inflammatory cytokines.

Development of Bifunctional Three-Dimensional Cysts from Chemically Induced Liver Progenitors.

Chemically induced liver progenitors (CLiPs) have promising applications in liver regenerative medicine. Three-dimensional (3D) structures generated from liver progenitor cells possess wide applications in cell transplantation, disease model, and drug testing. Here, we report on the spontaneous formation of 3D cystic structures comprising maturing rat CLiPs on gelatin-coated dishes. Our 3D cysts contained Alb+/+CK19+/- and Ck19+/+Alb+/- cells. These cell types gradually diverged into specialized mature cells, as demonstrated by the expression of mature biliary markers (Cftr, Ae2, and Aqp1) and hepatic markers (Alb and Mrp2). The 3D cysts also expressed functional multidrug resistance protein 1 (Mdr1), as indicated by epithelial efflux of rhodamine. Furthermore, we observed bile canaliculi functions between hepatocytes and cholyl-lysyl-fluorescein extrusions, indicating that the functional characteristics of 3D cysts and active bile salt export pump (Bsep) transporters were intact. Thus, our study revealed a natural characteristic of rat CLiPs to spontaneously form 3D cystic structures accompanied with cell maturation in vitro, offering a platform for studies of liver development and drug screening.

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.

Interleukin-12 prevents colorectal cancer liver metastases in mice.

Interleukin (IL)-12 has emerged to be a prospective molecule for antitumor therapies with many types of cancers. Here we examine the effect of IL-12 treatment in preventing the colorectal cancer liver metastasis in a mice model. At different doses, we found that IL-12 treatment decreased the formation of liver metastasis sites and the percentage of metastasis volume in the liver. Additionally, this treatment leads to improved survival function of mice after tumor cell transplantation. We believe that IL-12 based therapy provided a novel treatment to colorectal cancer patients suffering from liver metastasis.

Clinical experience in diagnosis and treatment of malignant gastrointestinal stromal tumors.

This study investigated the clinical pathologic character of malignant gastrointestinal stromal tumors (MGIST), their treatment with surgery, and evaluated the efficacy of imatinib postoperation. A total of 68 MGIST patients were enrolled. Of these, 27 patients underwent imatinib auxiliary therapy (treatment group) and 41 underwent imatinib therapy (control group). The therapeutic effects on the two groups were compared using χ(2) test analysis after follow-up of two years. The expressions of CD117, CD34, S100, Vimentin, and alpha smooth-muscle actin (SMA) were detected by immunohistochemistry methods. Of the 68 cases, 28 showed potential MGIST, whereas 40 had MGIST. Haemorrhagia or necrosis, abundant cell, manifest heteromorphism, and caryocinesia were observed in varying degrees. The positive rates of CD117, CD34, Vimentin, S100, and SMA were 89.7% (61/62), 88.2% (60/62), 73.5% (50/62), 41.1% (28/62) and 25.0% (17/62), respectively. The recurrence rate in the treatment group was significantly lower than that in the control group (p < 0.01). We concluded that CD117 and CD34 may be the most valuable markers in the diagnosis of MGIST, and the diagnosis of MGIST depends on the pathology. Surgery is a far better approach in the treatment of such patients, and imatinib is the more efficient target drug in preventing recurrence and metastasis.

[Prospective study on the use of nickel-titanium temperature-dependent memory-shape device (CAR27) for anastomosis after colorectal surgery].

OBJECTIVE: To prospectively evaluate the safety and efficacy of nickel- titanium temperature-dependent memory-shape device(CAR27) for colorectal anastomosis. METHODS: Sixty colorectal cancer patients were randomly divided into two groups and received colorectal anastomosis with CAR27 or traditional stapling device. Complications, bowel function return, and the extrusion of anastomosis ring were prospectively monitored. RESULTS: Both CAR27 and stapler group had one case of anastomotic leakage. Other complications such as stricture or obstruction were not found. Time for anastomosis of the two groups were (10.1±1.2) minutes and (11.2±2.1) minutes respectively. Time to first flatus was(3.2±1.2) days and (3.5±1.4) days respectively. Time to food intake resumption was (4.0±1.4) days and (4.3±1.3) days respectively. The differences above between the two groups were not statistically significant(P>0.05). The ring was expelled with stool within 7-16 days. The two groups were similar in operative time and the return of bowel function. CONCLUSION: CAR27 is safe and simple for colorectal anastomosis.

[Preparation of doxorubicin encapsulated in amphiphilic polysaccharide nanoparticles and anti-hepatocarcinoma effect thereof].

OBJECTIVE: To investigate the sustained release rule of doxorubicin/polylactide-grafted dextran copolymer (DOX/DEX-PLA) nanoparticles and the effect thereof in killing hepatocarcinoma cells. METHODS: DOX/DEX-PLA nanoparticles were prepared by method of emulsification & evaporation of organic solvent. Its morphology was observed by transmission electron microscopy and the encapsulating efficiency of DOX was determined by ultraviolet spectrophotometry. DOX/DEX-PLA was put in a dialysis bag to observe the releasing characteristics of DOX from DOX/DEX-PLA nanoparticles in vitro. Human liver carcinoma cells of the line HepG2 were cultured with DOX/DEX-PLA of different concentrations or the original drug of DOX as control group for 24, 36, or 48 h. MTT method was used to observe the cancer inhibition rate. BALB/c nude mice underwent subcutaneous injection of HepG2 cells at the right scapula and then randomly divided into 4 groups to undergo intravenous injection of DOX/DEX-PLA (excremental group), original drug of DOX (naked drug group), DEX-PLA (nanovector group), or normal saline (blank control group) once every 5 days for 3 times. Twenty-one days later the mice were killed with the tumors taken out to measure the weight to analyze the inhibitory effect against hepatocarcinoma cells. RESULTS: The DOX/DEX-PLA nanoparticles were of round or elliptical shape with the diameter of about 83 nm, and the DOX entrapment efficiency was about 67.1%. The releasing test in vitro manifested a sustained release of over 50% of DOX encapsulated in DOX/DEX-PLA nanoparticles for about 7 days. Both the DOX/DEX-PLA nanoparticles and naked drug DOX inhibited the growth of HepG2 cells with a similar inhibitory rate (51.3% vs 50.7%, P > 0.05), meanwhile the DEX-PLA nanovector failed to inhibit the HepG2 cells. In-vivo experiment showed an inhibitory rate of DOX/DEX-PLA nanoparticles on hepatocellular carcinoma xenografts of 68.56%, significantly higher than that of the naked drug DOX (48.17%). CONCLUSION: DOX/DEX-PLA nanoparticles can effectively inhibit the growth of hepatocarcinoma cells.

[Protection against liver injuries induced by low-temperature preservation with ulinastatin].

OBJECTIVE: To study whether ulinastatin has a protective role in cold preservation of liver and its mechanism. METHODS: To study the role of ulinastatin in protection of liver after cold preservation, the isolated rat liver with perfused either with cold perfusion fluid or perfused with cold perfusion fluid with ulinastatin. Liver tissue was harvested at 0, 12 and 24 hours after cold perfusion, and morphological changes were examined, alanine aminotransferase (ALT), lactate dehydrogenase (LDH), myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD) contents were also determined. RESULTS: ALT, LDH, MPO and MDA contents were significantly lower in ulinastatin group than those in the control group at 12 and 24 hours, and levels of ALT, LDH, MDA and MPO were increased along with prolongation of preservation time (all P<0.01), but SOD was higher in ulinastatin group than the control group and it was increased along with the prolongation of time (all P<0.01). Compared with ulinastatin group, the control group showed obvious pathological changes. CONCLUSION: Protective effect of UW solution used in clinic weakens gradually as cold preservation time is prolonged. Ulinastatin has protective effect on liver against cold preservation injury, and its effect is better than the simple UW solution. Its mechanism is related to inhibition of anti-oxidation and aggregation neutrophil's accumulation effect of ulinastatin.