[Investigating the effects of Modified Sijunzi Decoction on the diversity of intestinal microflora of severe scald rabbits based on 16S ribosomal RNA high-throughput sequencing].

Objective: To investigate the effects of Modified Sijunzi Decoction on the diversity of intestinal microflora of in severe scald rabbits based on 16S ribosomal RNA (16S rRNA) high-throughput sequencing. Methods: The experimental research method was adopted. Ninety Japanese big-ear rabbits regardless gender, aged 6 to 8 months, were randomly divided into normal control group, scald alone group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group, with 18 rabbits in each group. The rabbits in normal control group were free to eat and drink, and the rabbits in scald alone group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group were intragastrically administered normal saline, 0.2 g/mL Modified Sijunzi Decoction, 1.0 g/mL Modified Sijunzi Decoction, and 5.0 g/mL Modified Sijunzi Decoction, respectively for 7 days after sustaining full-thickness scalding of 30% total body surface area. On the 1st, 3rd, and 7th day after grouping, the levels of tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and IL-10 in ileal mucosa tissue of rabbits in each group were determined by enzyme-linked immunosorbent assay, and the number of samples in each group at each time point was 6. According to the above experimental results, another 9 rabbits were selected and divided into normal control group, scald alone group and scald+medium-dose group, with 3 rabbits in each group. The grouping and treatment methods of rabbits in each group were the same as before. On the 7th day after grouping, the V3, V4 region of 16S rRNA of ileum mucosa of rabbits in three groups were sequenced by high-throughput sequencing technology. The number of quality bacteria was counted by QIME software. The classifications of phylum, class, order, family and genus of microflora were analyzed by RDP Classifier software. The α diversity (Ace, Chao1, Simpson, and Shannon indexes) and ß diversity were analyzed by Illumina MiSeq sequencing technology, and the number of experiment samples in each group was 3. Data were statistically analyzed with analysis for variance of factorial design, SNK test, and Bonferroni correction. Results: Compared with that in normal control group, the levels of TNF-α of ileal mucosa tissue of rabbits in scald alone group, scald+low-dose group, and scald+high-dose group on the 1st, 3rd, and 7th day after grouping and scald+medium-dose group on the 1st and 3rd day after grouping were all significantly increased (P<0.01), the levels of IL-1ß in ileal mucosa tissue of rabbits in scald alone group, scald+low-dose group, scald+medium-dose group and scald+high-dose group on the 1st, 3rd, and 7th day after grouping were all significantly increased (P<0.05 or P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald alone group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 1st, 3rd, and 7th day after grouping were all significantly decreased (P<0.01). Compared with that in scald alone group, the levels of TNF-α in ileal mucosa tissue of rabbits in scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 3rd and 7th day after grouping, and scald+medium-dose group on the 1st day after grouping were all significantly decreased (P<0.01), and the levels of IL-1ß in ileal mucosa tissue of rabbits in scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 3rd and 7th day after grouping and scald+medium-dose group on the 1st day after grouping were all significantly decreased (P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald+low-dose group on the 7th day after grouping and scald+medium-dose group on the 1st, 3rd, and 7th day after grouping and scald+high-dose group on the 3rd and 7th day after grouping were all significantly increased (P<0.05 or P<0.01). Compared with that in scald+low-dose group, the levels of TNF-α in ileal mucosa tissue of rabbits in medium-dose scald alone group on the 1st, 3rd, and 7th day after grouping and in high-dose scald alone group on the 3rd and 7th day after grouping were significantly decreased (P<0.01), and the levels of IL-1ß in ileal mucosa tissue of rabbits in medium-dose scald alone group on the 1st, 3rd, and 7th day after grouping and in high-dose scald alone group on the 3rd and 7th day after grouping were all significantly decreased (P<0.05 or P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald+medium-dose group on the 1st, 3rd, and 7th day after grouping and in scald+high-dose group on the 7th day after grouping were all significantly increased (P<0.05 or P<0.01). Compared with that in scald medium-dose group, the levels of TNF-α in ileal mucosa tissue of rabbits in scald+high-dose group on the 1st, 3rd, and 7th day after grouping were all significantly increased (P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald+high-dose group on the 1st, 3rd, and 7th day after grouping were all significantly decreased (P<0.01), and the levels of IL-1ß in ileal mucosa tissue of rabbits in scald+high-dose group on the 7th day after grouping was significantly decreased (P<0.01). Compared with that on the 1st day after grouping, the levels of TNF-α in ileal mucosa tissue of rabbits in scald alone group on the 3rd and 7th day after grouping and in normal control group on the 3rd day after grouping were all significantly increased (P<0.05 or P<0.01), and the levels of IL-1ß in ileal mucosa tissue of rabbits in scald alone group both on the 3rd and 7th day after grouping were significantly increased (P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in both scald+low-dose group and scald+high-dose group on the 7th day after grouping and scald+medium-dose group both on the 3rd and 7th day after grouping were significantly increased (P<0.05 or P<0.01), and the levels of TNF-α in ileal mucosa tissue of rabbits in scald+high-dose group on the 3rd and 7th day after grouping and in scald+medium-dose group on the 7th day after grouping were all significantly decreased (P<0.05 or P<0.01), and the level of IL-1ß in ileal mucosa tissue of rabbits in scald+medium-dose group on the 7th day after grouping was significantly decreased (P<0.01), and the level of IL-10 in ileal mucosa tissue of rabbits in scald alone group on the 7th day after grouping was significantly decreased (P<0.01). Compared with that on the 3rd day after grouping, the levels of TNF-α and IL-1ß in ileal mucosa tissue of rabbits in scald alone group and the levels of IL-10 in ileal mucosa tissue of rabbits in normal control group, scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 7th day after grouping were all significantly increased (P<0.05 or P<0.01); and the levels of TNF-α in ileal mucosa tissue of rabbits in scald+low-dose group, scald+medium-dose group, and scald+high-dose group on the 7th day after grouping were all significantly decreased (P<0.05), and the levels of IL-1ß in ileal mucosa tissue of rabbits both in scald+medium-dose group and scald+high-dose group on the 7th day after grouping were significantly decreased (P<0.05 or P<0.01), and the levels of IL-10 in ileal mucosa tissue of rabbits in scald alone group on the 7th day after grouping was significantly decreased (P<0.01). On the 7th day after grouping, the high-quality sequences obtained from the microflora in ileum mucosa of rabbits in normal control group, scald alone group, and scald+medium-dose group were 96 023, 107 365, and 95 921, respectively. At the classification level of phylum, class, order, family, and genus of the microflora in ileum mucosa of rabbits in three groups were all Bacteroidetes and Firmicutes, Clostridium and Bacteroidetes, Clostridium and Bacteroidetes, Rumenobacteriaceae and Clostridium and Bacteroideaceae, Clostridium and Bacteroidetes and rumen bacteria mainly, while the percentage of microflora in each group was different. There were no significant differences in Ace, Chao1, Simpson, Shannon indices (P>0.05), and no obvious difference in ß diversity of microflora in ileal mucosa tissue of rabbits among three groups. Conclusions: After severe scalding, the inflammatory response of rabbit ileal mucosa tissue is obvious and increased in a time-dependent manner. Modified Sijunzi Decoction can reduce inflammation with optimal therapeutic concentration of 1.0 g/mL. The technology of high-throughput sequencing can reflect the structural composition of the intestinal microflora accurately. The ileal microflora of the severe scald rabbit can be regulated by the administration of Modified Sijunzi Decoction.

[Early efficacy of islet transplantation in the treatment of adult advanced diabetes].

Objective: To evaluate the safety and efficacy of islet transplantation for patients with advanced diabetes. Methods: Five cases of islet allotransplantation were performed on 4 adult recipients. The same blood type adult brain-dead pancreas donors were selected and the islets were prepared in GMP laboratory. The prepared islet suspension was slowly injected into the liver of the recipients within 30-60 minutes. The immunosuppressive regimen was a combination of basiliximab, tacrolimus and mycophenolate mofetil and TNF-alpha monoclonal antibody was used to reduce the post-transplant inflammatory response. Insulin was temporarily applied to control blood glucose after surgery, and the dosage of insulin was adjusted to decrease according to the blood glucose level until it was discontinued. Results: A total of 5 islet transplants were performed in 4 patients, including 1 patient who received the second islet transplantations. All operations were succeed and the blood glucose and portal pressure were stable during the operation. Exogenous insulin was continued to keep blood glucose level stable (4-12 mmol/L) after surgery. Four cases (including the one who received two islet transplantation) started to stop using insulin at the third to fourth week, and the insulin dosage of the other case was 74% lower than that before the operation, and no hypoglycemic reaction occurred in all patients after islet transplantation. The C-peptide level in 3 patients reached the normal range, and the level in one patient with type I diabetes (without insulin release) remained at 0.45-0.6 µg/L (0.15-0.2 nmol/L). In addition, one patient showed a rise in blood glucose again and continued to use insulin half a year after insulin discontinuation. Then, he was performed the second islet transplantation which showed good effect and stopped taking insulin in 10 days after surgery. There were 3 cases of liver puncture bleeding after opeation, of which 2 cases were treated with ultrasound radiofrequency ablation to stop bleeding, 1 case stopped spontaneously, and no other complications were found. Conclusions: Islet transplantation is effective in the treatment of advanced diabetes patients with small trauma and high safety, which is worthy of more promotion. Long-term efficacy and maintenance therapy still need further investigation.

Effects of miR-155 on proliferation and apoptosis by regulating FoxO3a/BIM in liver cancer cell line HCCLM3.

The article "Effects of miR-155 on proliferation and apoptosis by regulating FoxO3a/BIM in liver cancer cell line HCCLM3, by W.-W. Liao, C. Zhang, F.-R. Liu, W.-J. Wang, published in Eur Rev Med Pharmacol Sci 2018; 22 (5): 1277-1285. DOI: 10.26355/eurrev_201803_14468. PMID: 29565484." has been withdrawn from the authors. The Publisher apologizes for any inconvenience this may cause. https://www.europeanreview.org/article/14468.

[Effect of Wnt/ß-catenin signal regulating EMT level on the differentiation of mouse ESC to liver tissue].

Objective: To study embryonic stem cell (ESC) differentiation into liver tissue structure from the perspective of epithelial mesenchymal transition (EMT). Methods: ESC of Balb/c mice was selected to induced into hepatic cell using hepatocyte growth factor (HGF), fibroblast growth factor (FGF) in vitro, and at the time points of metaphase (13 d) and maturity (17 d) of differentiation, dynamic inhibition of Wnt/ß-catenin signal was made to reduce the level of EMT. Finally, three-dimensional organization structure growth of the differentiation cells was observed in the differentiation system.Expressions of the liver cells vascular markers[albumin (Alb) and vascular endothelial growth factor (VEGFR)]were detected. Results: During the differentiation of ESC, the level of early EMT in the experimental group and the control group was not significantly different. The level of mid-late EMT in the experiment group was significantly lower than the control group. On the day 18 and 20 of differentiation, the relative mRNA expression level of E-cadherin was 0.61±0.15 and 0.47±0.05 in the experimental group, and 0.07±0.05 and 0 in the control group, respectively.The expression level of ALB/AFP/CK8/CK19 in the experimental group was generally higher than that of the control group in the same period, while CD31/VEGFR1 markers in the experimental group decreased more slowly in the late period of differentiation compared with the control group. In the supernatant of ESC culture, the Alb of the experimental group could be detected onday 7, and the concentration was (0.32±0.02) mg/L, while Alb in the control group was (0.19±0.05) mg/L. Urea in the experimental group could be detected on the day 13, and the concentration was (8.7 ±1.0) µmol/L, and the urea concentration of the control group was (3.1±1.2) µmol/L. The concentration of Alb and urea in the culture supernatant of ESC differentiation system increased significantly with the prolongation of the differentiation time, and the Alb and urea concentrations in the experimental group were significantly higher than those in the control group at the same time period. In addition, the differentiated cells in the experimental group could maintain the growth of three-dimensional tissue, while the differentiated cells in the control group eventually showed a single cell state. The expression of hepatic and vascular cell markers could be detected in the experimental group. Immunofluorescence results showed that the hepatocytes and vascular structures were tightly arranged. HE staining showed the formation of hepatic lobular structure, while the control group had no vascular component markers. Conclusion: The differentiation of ESC into liver tissue can be effectively promoted by decreasing the level of EMT at the mid-late stage of ESC differentiation.

Effects of miR-155 on proliferation and apoptosis by regulating FoxO3a/BIM in liver cancer cell line HCCLM3.

OBJECTIVE: MiR-155 has been shown to be up-regulated in hepatocellular carcinoma (HCC) patients. B-cell lymphoma-2 (Bcl-2) interacting mediator of cell death (BIM) regulates cell proliferation and apoptosis, as its down-regulation is involved in HCC onset. Transcriptional factor FoxO3a mediates BIM expression and is related to HCC pathogenesis. Bioinformatics analysis showed targeted regulation of FoxO3a by miR-155. This study aims to investigate whether miR-155 plays a role in mediating FoxO3a/BIM signal pathway and HCC occurrence. PATIENTS AND METHODS: HCC patients were collected for tumor and adjacent tissues, in which microRNA-155 (miR-155) and FoxO3a expressions were examined. In vitro cultured HCCLM3, HepG2 and L-02 cells were tested for basal apoptotic rate by flow cytometry and were compared for miR-155 and FoxO3a expression. Dual-luciferase reporter gene assay demonstrated the targeted relationship between miR-155 and FoxO3a. HCCLM3 cells were treated with miR-155 inhibitor and/or FoxO3a-pMD18-T. Cell apoptosis and proliferation were examined by using flow cytometry and MTT assays, respectively. Western blot and spectrometry assay were employed to quantify the FoxO3a, BIM expressions, and caspase activity. RESULTS: Compared to adjacent tissues, HCC tissues had significantly higher miR-155 and significantly lower FoxO3a expression (p<0.05). HCCLM3 and HepG2 cells had significantly lower FoxO3a expression and basal apoptotic rate compared to L02 cells, whilst miR-155 level was significantly higher (p<0.05). MiR-155 targeted and inhibited 3'-UTR of FoxO3a, increasing BIM expression, caspase-3, and caspase-9 activities, and enhancing cell apoptosis and weakening proliferation. CONCLUSIONS: HCC tissues elevated the miR-155 and suppressed the FoxO3a expressions. MiR-155 targeted and inhibited FoxO3a expression to suppress the BIM, depress caspase-3 and caspase-9 activities, therefore inhibiting the HCC cell apoptosis and facilitating proliferation.

[The effect of semimature dendritic cell and the levels of Treg on transplantation tolerance of hepatocytes differentiated from mouse embryonic stem cell].

Objective: To investigate the inducing effect and mechanism of semimature dendritic cell (smDCs) on transplantation tolerance of hepatocytes differentiated from mouse embryonic stem cells (ESCs), and to study the connections between smDCs and regulatory dendritic cells (regDCs). Methods: ESCs of 129 mouse labelled green fluorescent protein (GFP) were induced to hepatocytes by using previous methods. Meanwhile, bone marrow mononuclear cells of 129 mouse were induced to smDCs and regDCs. Moreover, the hepatocytes differentiated from 129 mouse ESCs were transplanted into liver of BALB/c mouse 3 days after infusing smDCs and regDCs suspension of 129 mouse into BALB/c mouse by tail vein respectively. After that, the growth status and survival time of transplanted cells in the recipient and infiltration of lymphocytes in transplant sites were observed. Furthermore, Foxp3 expression of peripheral blood CD4+ T cells was also tested. Results: In the control group, the transplanted cells in liver of BALB/c mouse survived only about 1 week. In contrast, the transplanted cells of smDC groups and regDCs groups survived about 4 weeks and the transplant sites of smDC groups also had less CD3(+) T cells. The morphology of smDCs were similar with regDCs. The expression of MHC-Ⅱ, CD40, CD80 and CD86 on smDCs and regDCs were moderate. Moreover, the Foxp3 expression of peripheral blood CD4+ T cells in smDC groups was higher than that in the control groups, from 1.11% up to 5.38%. The Foxp3 expression in regDC groups rose to 3.87%. Conclusion: The smDCs could induce transplantation tolerance of hepatocytes differentiated from 129 mouse ESCs in the recipient. The mechanism was associated with high level of Foxp3(+) Tregs, which could be increased by means of smDCs appropriate expression of MHC-Ⅱ, CD40, CD80 and CD86. The smDCs and regDCs were the same type of tolerance dendritic cells.