Zhigang Ren

Application of metagenomics in the human gut microbiome

There are more than 1000 microbial species living in the complex human intestine. The gut microbial community plays an important role in protecting the host against pathogenic microbes, modulating immunity, regulating metabolic processes, and is even regarded as an endocrine organ. However, traditional culture methods are very limited for identifying microbes. With the application of molecular biologic technology in the field of the intestinal microbiome, especially metagenomic sequencing of the next-generation sequencing technology, progress has been made in the study of the human intestinal microbiome. Metagenomics can be used to study intestinal microbiome diversity and dysbiosis, as well as its relationship to health and disease. Moreover, functional metagenomics can identify novel functional genes, microbial pathways, antibiotic resistance genes, functional dysbiosis of the intestinal microbiome, and determine interactions and co-evolution between microbiota and host, though there are still some limitations. Metatranscriptomics, metaproteomics and metabolomics represent enormous complements to the understanding of the human gut microbiome. This review aims to demonstrate that metagenomics can be a powerful tool in studying the human gut microbiome with encouraging prospects. The limitations of metagenomics to be overcome are also discussed. Metatranscriptomics, metaproteomics and metabolomics in relation to the study of the human gut microbiome are also briefly discussed.

Intestinal Microbial Variation May Predict Early Acute Rejection after Liver Transplantation in Rats

Background Acute rejection (AR) remains a life-threatening complication after orthotopic liver transplantation (OLT) and there are few available diagnostic biomarkers clinically for AR. This study aims to identify intestinal microbial profile and explore potential application of microbial profile as a biomarker for AR after OLT. Methods The OLT models in rats were established. Hepatic graft histology, ultrastructure, function, and intestinal barrier function were tested. Ileocecal contents were collected for intestinal microbial analysis. Results Hepatic graft suffered from the ischemia-reperfusion (I/R) injury on day 1, initial AR on day 3, and severe AR on day 7 after OLT. Real-time quantitative polymerase chain reaction results showed that genus Faecalibacterium prausnitzii and Lactobacillus were decreased, whereas Clostridium bolteae was increased during AR. Notably, cluster analysis of denaturing gradient gel electrophoresis (DGGE) profiles showed the 7AR and 3AR groups clustered together with 73.4% similarity, suggesting that intestinal microbiota was more sensitive than hepatic function in responding to AR. Microbial diversity and species richness were decreased during AR. Phylogenetic tree analysis showed that most of the decreased key bacteria belonged to phylum Firmicutes, whereas increased key bacteria belonged to phylum Bacteroidetes. Moreover, intestinal microvilli loss and tight junction damage were noted, and intestinal barrier dysfunction during AR presented a decrease of fecal secretory immunoglobulin A (sIgA) and increase of blood bacteremia, endotoxin, and tumor necrosis factor-&agr;. Conclusion We dynamically detail intestinal microbial characterization and find a high sensitivity of microbial change during AR after OLT, suggesting that intestinal microbial variation may predict AR in early phase and become an assistant therapeutic target to improve rejection after OLT.

Protective effect of probiotics on intestinal barrier function in malnourished rats after liver transplantation

BACKGROUND Most patients waiting for liver transplantation have end-stage liver diseases with malnutrition, which is prone to induce intestinal barrier dysfunction after liver transplantation. We aimed to study the effect of probiotics on intestinal barrier function in malnourished rats following liver transplantation with long-term antibiotics. METHODS Twelve Lewis rats were selected as donors. Twelve BN rats, which served as recipients, were subjected to malnutrition by semi-starvation for 4-5 weeks. They were randomly divided into two groups: a control group which received phosphate-buffered saline and a probiotics group which received Bifidobacterium and Lactobacillus. All recipients were injected with intramuscular imipenem and subcutaneous cyclosporine A. Furthermore, six normal BN rats without any drugs or operations served as a normal group. Eight days after operation, all rats were sacrificed for examination of the following parameters: serum levels of endotoxin and TNF-alpha, bacterial translocation, intestinal microflora, ileocecal sIgA, lymphocyte numbers, and phenotypes (CD4, CD8, alphabetaTCR, gammadeltaTCR) of Peyers patches. RESULTS In recipients subjected to malnutrition, weight decreased by 20% and they survived until 8 days after operation. Compared with the normal group, all recipients on postoperative day 8 showed increased levels of serum endotoxin and TNF-alpha as well as increased counts of translocated bacteria. Meanwhile, there were decreases in counts of Bifidobacterium and Lactobacillus in the ileocecum, sIgA concentration, and lymphocytes of Peyers patches. Moreover, partial alteration in lymphocyte phenotypes was evidenced by elevated ratios of CD8+ and gammadeltaTCR+ lymphocytes. In contrast, compared to the control group, supplementation with probiotics reduced the levels of serum endotoxin, TNF-alpha and bacterial translocation, increased the counts of Bifidobacterium and Lactobacillus, the concentration of sIgA and lymphocytes of Peyers patches, and also slightly restored the alteration of lymphocyte phenotypes. CONCLUSION Supplementation with probiotics including Bifidobac-terium and Lactobacillus promoted partial restoration of intestinal microflora and improved intestinal barrier function in malnourished rats after liver transplantation with long-term use of antibiotics.

Liver Ischemic Preconditioning (IPC) Improves Intestinal Microbiota Following Liver Transplantation in Rats through 16s rDNA-Based Analysis of Microbial Structure Shift

Background Ischemia-reperfusion (I/R) injury is associated with intestinal microbial dysbiosis. The “gut-liver axis” closely links gut function and liver function in health and disease. Ischemic preconditioning (IPC) has been proven to reduce I/R injury in the surgery. This study aims to explore the effect of IPC on intestinal microbiota and to analyze characteristics of microbial structure shift following liver transplantation (LT). Methods The LT animal models of liver and gut IPC were established. Hepatic graft function was assessed by histology and serum ALT/AST. Intestinal barrier function was evaluated by mucosal ultrastructure, serum endotoxin, bacterial translocation, fecal sIgA content and serum TNF-α. Intestinal bacterial populations were determined by quantitative PCR. Microbial composition was characterized by DGGE and specific bacterial species were determined by sequence analysis. Principal Findings Liver IPC improved hepatic graft function expressed as ameliorated graft structure and reduced ALT/AST levels. After administration of liver IPC, intestinal mucosal ultrastructure improved, serum endotoxin and bacterial translocation mildly decreased, fecal sIgA content increased, and serum TNF-α decreased. Moreover, liver IPC promoted microbial restorations mainly through restoring Bifidobacterium spp., Clostridium clusters XI and Clostridium cluster XIVab on bacterial genus level. DGGE profiles indicated that liver IPC increased microbial diversity and species richness, and cluster analysis demonstrated that microbial structures were similar and clustered together between the NC group and Liver-IPC group. Furthermore, the phylogenetic tree of band sequences showed key bacteria corresponding to 10 key band classes of microbial structure shift induced by liver IPC, most of which were assigned to Bacteroidetes phylum. Conclusion Liver IPC cannot only improve hepatic graft function and intestinal barrier function, but also promote restorations of intestinal microbiota following LT, which may further benefit hepatic graft by positive feedback of the “gut-liver axis”.

Nanosecond Pulsed Electric Field Inhibits Cancer Growth Followed by Alteration in Expressions of NF-κB and Wnt/β-Catenin Signaling Molecules

Cancer remains a leading cause of death worldwide and total number of cases globally is increasing. Novel treatment strategies are therefore desperately required for radical treatment of cancers and long survival of patients. A new technology using high pulsed electric field has emerged from military application into biology and medicine by applying nsPEF as a means to inhibit cancer. However, molecular mechanisms of nsPEF on tumors or cancers are still unclear. In this paper, we found that nsPEF had extensive biological effects in cancers, and clarified its possible molecular mechanisms in vitro and in vivo. It could not only induce cell apoptosis via dependent-mitochondria intrinsic apoptosis pathway that was triggered by imbalance of anti- or pro-apoptosis Bcl-2 family proteins, but also inhibit cell proliferation through repressing NF-κB signaling pathway to reduce expressions of cyclin proteins. Moreover, nsPEF could also inactivate metastasis and invasion in cancer cells by suppressing Wnt/β-Catenin signaling pathway to down-regulating expressions of VEGF and MMPs family proteins. More importantly, nsPEF could function safely and effectively as an anti-cancer therapy through inducing tumor cell apoptosis, destroying tumor microenvironment, and depressing angiogenesis in tumor tissue in vivo. These findings may provide a creative and effective therapeutic strategy for cancers.

Enteral supplementation with glycyl-glutamine improves intestinal barrier function after liver transplantation in rats

BACKGROUND Most patients after liver transplantation (LT) suffer from intestinal barrier dysfunction. Glycyl-glutamine (Gly-Gln) by parenteral supplementation is hydrolyzed to release glutamine, which improves intestinal barrier function in intestinal injury. This study aimed to investigate the effect of Gly-Gln by enteral supplementation on intestinal barrier function in rats after allogenetic LT under immunosuppressive therapy. METHODS Twelve inbred Lewis rats were selected randomly as donors, and 24 inbred Brown Norway (BN) rats as recipients of allogenetic LT. The recipients were divided into a control group (Ala, n=12) and an experimental group (Gly-Gln, n=12). In each group, 6 normal BN rats were sampled for normal parameters on preoperative day 3. The 6 recipients in the control group received alanine (Ala) daily by gastric perfusion for 3 preoperative days and 7 postoperative days, and the 6 recipients in the experimental group were given Gly-Gln in the same manner. The 12 BN recipients underwent orthotopic LT under sterile conditions after a 3-day fast and were given immunosuppressive therapy for 7 days. They were harvested for sampling on postoperative day 8. The following parameters were assessed: intestinal mucosal protein content, mucosal ultrastructure, ileocecal sIgA content, portal plasma levels of endotoxin and TNF-alpha, and bacterial translocation. RESULTS All recipients were alive after LT. On preoperative day 3, all parameters were similar in the two groups. On postoperative day 8, all parameters in the two groups were remarkably changed from those on preoperative day 3. However, compared to the Ala group, supplementation with Gly-Gln increased the levels of intestinal mucosal protein and ileocecal sIgA, improved mucosal microvilli, and decreased portal plasma levels of endotoxin and TNF-alpha as well as bacterial translocation. CONCLUSION Enteral supplementation with Gly-Gln improved intestinal barrier function after allogenetic LT in rats.

MRC-5 fibroblast-conditioned medium influences multiple pathways regulating invasion, migration, proliferation, and apoptosis in hepatocellular carcinoma

BackgroundCarcinoma associated fibroblasts (CAFs), an important component of tumor microenvironment, are capable of enhancing tumor cells invasion and migration through initiation of epithelial–mesenchymal transition (EMT). MRC-5 fibroblasts are one of the CAFs expressing alpha-smooth muscle actin. It is ascertained that medium conditioned by MRC-5 fibroblasts stimulate motility and invasion of breast cancer cells. However, its role in hepatocellular carcinoma (HCC) is less clear. The aim of our study was to investigate the effect of MRC-5-CM on HCC and explore the underlying mechanisms.Methods and resultsUsing a combination of techniques, the role of MRC-5-CM in HCC was evaluated. We determined that MRC-5-CM induced the non-classical EMT in Bel-7402 and MHCC-LM3 cell lines. Initiation of the non-classical EMT was mainly via quintessential redistribution of α-, β- and γ-catenin, P120 catenin, E-cadherin, and N-cadherin, rather than up-regulation of typical EMT-related transcription factors (i.e., Snail, Twist1, ZEB-1 and ZEB2). We also found that MRC-5-CM potentiated both the migration and invasion of Bel-7402 and MHCC-LM3 cells in mesenchymal movement mode through activation of the α6, β3, β4, β7 integrin/FAK pathway and upregulation of MMP2. The flow cytometric analysis showed that MRC-5-CM induced G1 phase arrest in Bel-7402 cells with a concomitant reduction of S phase cells. In contrast, MRC-5-CM induced S phase arrest in MHCC-LM3 cells with a concomitant reduction of cells in the G2/M phase. MRC-5-CM also inhibited apoptosis in Bel-7402 cells while inducing apoptosis in MHCC-LM3 cells.ConclusionCollectively, MRC-5-CM promoted HCC cell motility and invasiveness through initiation of the non-classical EMT, including redistribution of α-, β- and γ-catenin, P120 catenin, E-cadherin, and N-cadherin, activation of the integrin/FAK pathway, and upregulation of MMP2. Hence, MRC-5-CM exerted distinct roles in Bel-7402 and MHCC-LM3 cell viability by regulating cyclins, cyclin dependent kinases (CDKs), CDK inhibitors (CKIs), Bcl-2 family proteins and other unknown mechanosensors.

Laparoscopic resection of benign schwannoma in the hepatoduodenal ligament: A case report and review of the literature

Schwannomas are mesenchymal neoplasms with a low malignant potential, which arise from Schwann cells. The tumors can occur in most parts of the body; however, the head, neck and flexor surfaces of the extremities are the most common locations. Schwannomas occurring in the hepatoduodenal ligament are extremely rare. To the best of our knowledge, only two cases of schwannoma in the hepatoduodenal ligament have been reported in the literature, and treatment of such cases by laparoscopic surgery has not yet been reported. The present study reports a case of schwannoma in the hepatoduodenal ligament in a 50-year-old male patient. Physical and laboratory examinations showed no abnormal results. Ultrasound and computed tomography failed to definitively diagnose the mass and identify its location. During laparoscopic surgery, a mass was identified in the hepatoduodenal ligament and was completely removed. The gross specimen was a 4.5×2.5×2.5-cm localized mass, yellowish-white in color. Microscopic examination revealed that the tumor was mainly composed of spindle-shaped cells and no atypical cells were identified. Immunohistochemical staining showed a strong positive S-100 protein reaction, whereas cluster of differentiation 34 and epithelial membrane antigen were negative. The final diagnosis of the lesion was benign schwannoma of the hepatoduodenal ligament. The patient was followed-up for 7 months and, at the time of writing, was healthy and without any complications. The aim of the present study was to describe a rare case of hepatoduodenal ligament schwannoma in a 50-year-old male patient, and present a review of the literature. To the best of our knowledge, this is the first case of hepatoduodenal ligament schwannoma treated by laparoscopic surgery.

Clinical correlation of calpain-1 and glypican-3 expression with gallbladder carcinoma

Gallbladder carcinoma (GBC) possesses a poor prognosis, which is primarily attributed to the lack of early and timely surgical intervention. Calpain-1 and glypican-3 have been implicated in the progression of various types of cancer. The present study aimed to detect the expression of calpain-1 and glypican-3 in GBC, and analyzed whether the expression levels of these proteins correlated with any clinicopathological variables. A total of 100 patients with GBC and 30 patients with cholecystitis who accepted surgical treatment were enrolled in the present study. Pathological and clinical data were obtained from all patients. The expression of calpain-1 and glypican-3 was detected in paraffin-embedded tissues by immunohistochemistry. Calpain-1 expression was manually assessed with an immunohistochemical H-score with a slight modification. Glypican-3 expression was assessed as negative and positive. The correlations between protein expression and clinicopathological characteristics, and the associations between the proteins were analyzed. All patients exhibited positive expression of calpain-1. Notably, the high expression rate of calpain-1 was significantly increased in patients with GBC, compared with patients with cholecystitis (32.0 vs. 6.7%; χ2=7.668; P=0.006), suggesting that calpain-1 expression may be associated with progression from cholecystitis to GBC. In addition, the positive rate of glypican-3 expression was 53.0% in patients with GBC and 63.3% in patients with cholecystitis, with no significant difference (χ2=0.997; P=0.318). Furthermore, the expression of calpain-1 and glypican-3 had no significant correlation with gender, age, degree of tumor differentiation and tumor-node-metastasis classification in patients with GBC. Notably, the expression of calpain-1 and glypican-3 displayed a significant positive correlation in patients with GBC (r=0.517; P<0.01), but a significantly negative correlation (r=-0.856; P<0.01) in patients with cholecystitis. In conclusion, calpain-1 expression may be associated with progression from cholecystitis to GBC. Combined detection of calpain-1 and glypican-3 may be beneficial for prognosis assessment of GBC.

Chronic bile duct hyperplasia is a chronic graft dysfunction following liver transplantation

AIM To investigate pathological types and influential factors of chronic graft dysfunction (CGD) following liver transplantation (LT) in rats. METHODS The whole experiment was divided into three groups: (1) normal group (n = 12): normal BN rats without any drug or operation; (2) syngeneic transplant group (SGT of BN-BN, n = 12): both donors and recipients were BN rats; and (3) allogeneic transplant group (AGT of LEW-BN, n = 12): Donors were Lewis and recipients were BN rats. In the AGT group, all recipients were subcutaneously injected by Cyclosporin A after LT. Survival time was observed for 1 year. All the dying rats were sampled, biliary tract tissues were performed bacterial culture and liver tissues for histological study. Twenty-one day after LT, 8 rats were selected randomly in each group for sampling. Blood samples from caudal veins were collected for measurements of plasma endotoxin, cytokines and metabonomic analysis, and faeces were analyzed for intestinal microflora. RESULTS During the surgery of LT, no complications of blood vessels or bile duct happened, and all rats in each group were still alive in the next 2 wk. The long term observation revealed that a total of 8 rats in the SGT and AGT groups died of hepatic graft diseases, 5 rats in which died of chronic bile duct hyperplasia. Compared to the SGT and normal groups, survival ratio of rats significantly decreased in the AGT group (P < 0.01). Moreover, liver necrosis, liver infection, and severe chronic bile duct hyperplasia were observed in the AGT group by H and E stain. On 21 d after LT, compared with the normal group (25.38 ± 7.09 ng/L) and SGT group (33.12 ± 10.26 ng/L), plasma endotoxin in the AGT group was remarkably increased (142.86 ± 30.85 ng/L) (both P < 0.01). Plasma tumor necrosis factor-α and interleukin-6 were also significantly elevated in the AGT group (593.6 ± 171.67 pg/mL, 323.8 ± 68.30 pg/mL) vs the normal (225.5 ± 72.07 pg/mL, 114.6 ± 36.67 pg/mL) and SGT groups (321.3 ± 88.47 pg/mL, 205.2 ± 53.06 pg/mL) (P < 0.01). Furthermore, Bacterial cultures of bile duct tissues revealed that the rats close to death from the SGT and AGT groups were strongly positive, while those from the normal group were negative. The analysis of intestinal microflora was performed. Compared to the normal group (7.98 ± 0.92, 8.90 ± 1.44) and SGT group (8.51 ± 0.46, 9.43 ± 0.69), the numbers of Enterococcus and Enterobacteria in the AGT group (8.76 ± 1.93, 10.18 ± 1.64) were significantly increased (both P < 0.01). Meanwhile, compared to the normal group (9.62 ± 1.60, 9.93 ± 1.10) and SGT group (8.95 ± 0.04, 9.02 ± 1.14), the numbers of Bifidobacterium and Lactobacillus in the AGT group (7.83 ± 0.72, 8.87 ± 0.13) were remarkably reduced (both P < 0.01). In addition, metabonomics analysis showed that metabolic profiles of plasma in rats in the AGT group were severe deviated from the normal and SGT groups. CONCLUSION Chronic bile duct hyperplasia is a pathological type of CGD following LT in rats. The mechanism of this kind of CGD is associated with the alterations of inflammation, intestinal barrier function and microflora as well as plasma metabolic profiles.