Objective: To explore a simple and feasible method for the isolation and purification of hepatocytes, hepatic stellate cells (HSC), and lymphocytes from mice. Methods: The cell suspension was obtained from male C57bl/6 mice by hepatic perfusion through the portal vein digestion method and then isolated and purified by discontinuous Percoll gradient centrifugation. Trypan blue exclusion was used to determine cell viability. Glycogen staining, cytokeratin 18, and transmission electron microscopy were used to identify hepatic cells. Immunofluorescence was used to detect α-smooth muscle actin combined with desmin in HSCs. Flow cytometry was used to analyze lymphocyte subsets in the liver. Results: After isolation and purification, about 2.7×10(7) hepatocytes, 5.7×10(5) HSCS, and 4.6×106 hepatic mononuclear cells were obtained from the liver of mice with a body weight of about 22g. The cell survival rate in each group was > 95%. Hepatocytes were apparent in glycogen deposited purple-red granules and cytokeratin 18. Electron microscopy showed that there were abundant organelles in hepatocytes and tight junctions between cells. HSC had expressed α-smooth muscle actin and desmin. Flow cytometry showed hepatic mononuclear cells, including lymphocyte subsets such as CD4, CD8, NKs, and NKTs. Conclusion: The hepatic perfusion through the portal vein digestion method can isolate multiple primary cells from the liver of mice at once and has the features of simplicity and efficiency.
Male ; Mice ; Animals ; Keratin-18 ; Actins ; Desmin ; Liver ; Hepatocytes ; Hepatic Stellate Cells

Salvia plebeia has been in use as traditional Chinese medicine (TCM) for more than 500 years. In this study, the complete chloroplast (cp) genome of S. plebeia was sequenced, assembled and compared to those of other five published Salvia cp genomes. It was found that the cp genome structure of S. plebeia was well conserved and had a total size of 151 062 bp. Four parameters were used to display the usage conditions of the codons of the amino acids in Salvia genus. Although the number of protein-coding genes in each species was the same, the total number of codons was different. Except for amino acids Trp and Met whose Relative Synonymous Codon Usage (RSCU) value of one condon was equal to 1, the remaining 19 amino acids had 1-3 preferred codons. The preferred codon names of each amino acid were coincident. The period size for the tandem repeats of six species ranged from 9 to 410 bp. Salvia cp genomes mainly possessed tandem repeats with a copy number less than or equal to 3. The sequence length of tandem repeats of the six species ranged from 25 to 824 bp. Highly viarable regions including four intergenic spacers and six partial genes were discovered as potential specific barcodes for Salvia species through cp genome-wide comparison. Finally, we performed phylogenetic analyses based on the complete cp genome and coding sequences respectively. These results provide information to help construct the cp genome library for Salvia, which may support studies of phylogenetics, DNA barcoding, population and transplastomics.

BACKGROUND: Large-nerve fiber dysfunction, as assessed by vibration perception threshold (VPT) predicts risks of ulceration, amputation, and mortality in diabetes. Serum uric acid (UA) is closely associated with various metabolic disorders, especially diabetes. Thus, we sought to investigate the clinical relevance of UA to large-nerve fiber dysfunction, among patients with type 2 diabetes (T2D). METHODS: Medical records of consecutive patients with T2D who were admitted to Beijing Friendship Hospital Pinggu Campus between May 2014 and December 2016 were collected. Data for the 824 eligible patients included in the final analysis were extracted using a structured form. A VPT value ≥15 in either foot was defined as abnormal. We compared the clinical characteristics between patients with abnormal VPT and those with normal VPT (VPT value <15 in both feet) in the overall population and in gender subgroups. Logistic regression analysis was performed to explore the association of abnormal VPT with UA level. One-way analysis of variance was used to compare VPT values across four UA quartiles. RESULTS: UA levels were significantly lower in T2D patients with abnormal VPT than in those with normal VPT (294.5 ± 84.0 vs. 314.9 ± 92.8 μmol/L, P < 0.01), especially among male patients (311.7 ± 85.2 vs. 336.9 ± 89.6 μmol/L, P < 0.01). From the logistic regression analysis, hyperuricemia (males >420 μmol/L; females >360 μmol/L) was associated with a reduced risk of abnormal VPT (odds ratio [OR], 0.60; 95% confidence interval [CI], 0.39-0.91; P < 0.05). This association was robust in male patients (OR, 0.43; 95% CI, 0.24-0.76; P < 0.01) but not in female patients (OR, 0.92; 95% CI, 0.47-1.82; P = 0.816), even after adjustment for confounding factors. For the younger male subgroup (age <65 years), VPT values decreased as the UA level increased (P for trend = 0.002), but this trend was not significant in older male subgroup (age ≥65 years; P for trend = 0.400). CONCLUSIONS Low serum UA levels showed a significant association with an increased risk of large-nerve fiber dysfunction in male patients with T2D, but not in female patients with T2D. In addition, in only the younger subgroup of male patients (<65 years), lower levels of UA also correlated with higher VPT values.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Cross-Sectional Studies ; Diabetes Mellitus, Type 2 ; blood ; pathology ; Female ; Humans ; Male ; Middle Aged ; Nerve Fibers ; pathology ; Peripheral Nervous System Diseases ; blood ; pathology ; Uric Acid ; blood ; Young Adult

Background:: Large-nerve fiber dysfunction, as assessed by vibration perception threshold (VPT) predicts risks of ulceration, amputation, and mortality in diabetes. Serum uric acid (UA) is closely associated with various metabolic disorders, especially diabetes. Thus, we sought to investigate the clinical relevance of UA to large-nerve fiber dysfunction, among patients with type 2 diabetes (T2D). Methods:: Medical records of consecutive patients with T2D who were admitted to Beijing Friendship Hospital Pinggu Campus between May 2014 and December 2016 were collected. Data for the 824 eligible patients included in the final analysis were extracted using a structured form. A VPT value ≥15 in either foot was defined as abnormal. We compared the clinical characteristics between patients with abnormal VPT and those with normal VPT (VPT value <15 in both feet) in the overall population and in gender subgroups. Logistic regression analysis was performed to explore the association of abnormal VPT with UA level. One-way analysis of variance was used to compare VPT values across four UA quartiles. Results:: UA levels were significantly lower in T2D patients with abnormal VPT than in those with normal VPT (294.5 ± 84.0 vs. 314.9 ± 92.8 μmol/L, P < 0.01), especially among male patients (311.7 ± 85.2 vs. 336.9 ± 89.6 μmol/L, P < 0.01). From the logistic regression analysis, hyperuricemia (males >420 μmol/L; females >360 μmol/L) was associated with a reduced risk of abnormal VPT (odds ratio [OR], 0.60; 95% confidence interval [CI], 0.39–0.91; P < 0.05). This association was robust in male patients (OR, 0.43; 95% CI, 0.24–0.76; P < 0.01) but not in female patients (OR, 0.92; 95% CI, 0.47–1.82; P = 0.816), even after adjustment for confounding factors. For the younger male subgroup (age <65 years), VPT values decreased as the UA level increased (P for trend = 0.002), but this trend was not significant in older male subgroup (age ≥65 years; P for trend = 0.400). Conclusions: Low serum UA levels showed a significant association with an increased risk of large-nerve fiber dysfunction in male patients with T2D, but not in female patients with T2D. In addition, in only the younger subgroup of male patients (<65 years), lower levels of UA also correlated with higher VPT values.

OBJECTIVE Regulating P2x7R- NLRP3 inflammasome activation might be a potentialtherapeutic strategy to treat alcoholic hepatosteatosis. We investigated whether this process would be modulated by gentiopicroside (GPS), which is attributed to the bitterness of gentian root extract. METHODSAn in vivo model was established by intragastrically treating mice with ethanol, and an in vitro modelwas created by treating HepG2 cells with ethanol or treating RAW 264.7 macrophages and murinebone marrow- derived macrophages (BMDMs) with lipopolysaccharides (LPS) plus adenosine triphos-phate (ATP). RESULTS In alcoholic hepatosteatotic mice model, GPS decreased serum aminotrans-ferase and triglyceride accumulation. GPS regulated sterol regulatory element-binding protein-1 (Srebp1),peroxisome proliferators- actived receptors α (PPARα) and acetyl CoA carboxylase (ACC) expressionvia elevating liver kinase B1 (LKB1)/AMP-activated Kinase (AMPK). Suppression of nucleotide-bindingoligomerization domain-like receptor protein 3 (NLRP3), caspase-1 and expression by GPS resulted inthe inhibition of interleukin-1β (IL-1β) production. In ethanol-exposed HepG2 cells, GPS reduced lipo-genesis and promoted lipid oxidation via P2x7R- NLRP3 inflammasome activation. P2x7R silencingenhanced AMPK activity, and reduced Srebp1 expression in ethanol-treated hepatocytes. GPS down-regulated P2x7R-mediated inflammatory response to extracellular ATP in LPS-primed RAW 264.7 macro-phages and BMDMs. Additionally, P2x7R deficiency attenuated IL- 1β cleavage in RAW 264.7 macro-phages, and GPS further suppressed IL-1β cleavage. CONCLUSION Activation of LKB1/AMPK signalingby GPS might be mediated by P2x7R-NLRP3 inflammasome, suggesting a therapeutic utility of P2x7Rblockade in alcoholic hepatosteatosis treatment.

OBJECTIVE The current study was designed to investigate the anti-steatosis effect of Pleurotus citrinopileatus extract (PC) and the underlying mechanism in vivo and in vitro. METHODS Acute and chronic alcoholic hepatosteatosis murine models and ethanol-treated HepG2 cells were applied. RESULTS In vitro,the anti-steatosis effect of PC was further confirmed via Nile red staining in HepG2 cells treated with ethanol.Both of acute and chronic alcohol-induced mice hepatosteatosis model,PC decreased serum aminotransferase and triglyceride accumulation. Upregulated sterol-regulatory element binding protein1(Srebp1),purinergic ligand-gated ion channel 7receptor(P2X7R)and downregulated sirtuin1 (SIRT1), adenosine 5′-monophosphate (AMP)-activated protein kinase α (AMPKα) caused by acute and chronic alcohol intake were modulated by PC.In ethanol-exposed HepG2 cells,PC reduced lipid accumulation in a concentration-dependent manner and exhibited superior ability in controlling lipid accumulation compared with metformin. CONCLUSION PC could abolish hepatic lipid accumulation through regulating SIRT1-AMPKα signaling in acute and chronic alcohol-induced hepatic steatosis.

OBJECTIVE Dihydroquercetin(TAX)is the most abundant dihydroflavone found in on-ions,milk thistle and Douglas fir bark.We investigated whether TAX could inhibit the lipid accumulation in alcoholic liver steatosis in vivo and in vitro.METHODS An in vivo model was established by intragas-trically treating mice with ethanol,and an in vitro model was created by treating HepG2 cells with etha-nol.RESULTS TAX regulated Sterol Regulatory Element-binding Protein-1(SREBP1)and Acetyl CoA Carboxylase (ACC) expression via elevating Liver Kinase B1 (LKB1)/ AMP-activated Kinase (AMPK) phosphorylation. Also, TAX upregulated SIRT1 expression, which suppressed by ethanal intake. Suppression of Purinergic 2X7 receptor (P2x7R), nucleotide-binding oligomerization domain-like re-ceptor protein 3(NLRP3)and Cysteine protease-1(caspase-1)cleavage by TAX resulted in the inhibi-tion of Interleukin-1β(IL-1β) production and release. Additionally, TAX reduced lipogenesis and pro-moted lipid oxidation via the regulation of AMPK and ACC in ethanol-treated steatotic HepG2 cell.TAX downregulated IL-1β cleavage response to Lipopolysaccharides (LPS) plus adenosine triphosphate(ATP) stimulation in HepG2 cells. P2x7R deficiency attenuated lipid accumulation with increasing AMPK activity and decreasing SREBP1 expression in ethanol-treated HepG2 cells.CONCLUSION Our data showed that TAX exhibited the inhibitory properties on lipogenesis and hepatoprotective ca-pacity,indicating that TAX has therapeutic potential for preventing alcoholic liver steatosis.

Objective: To explore the efficacy and safety of recombinant human interleukin-11 (rhIL-11) in treatment of chemotherapy-induced thrombocytopenia of acute leukemia. Methods: Acute leukemia patients with chemotherapy-induced thrombocytopenia [Platelets (Plt) < 50×10⁹/L] in 6 centers nationwide from February 2016 to July 2016 were treated with rhIL-11 (2 mg/time, twice per day) by subcutaneous injection. Treatment lasted 7 days or at least until Plt≥ 50×10⁹/L. The Plt recovery was observed during treatment. Results: A total of 112 patients were enrolled, and 2 patients decided to drop out of study. The efficacy population consisted of 110 patients, and the total response rate reached 74.5% (82/110). The average variation of Plt during treatment was (70±54)×10⁹/L, and recovery average time of Plt for the patients with favorable efficacy was (8.7±3.0) days. In treatment with severe thrombocytopenia, rhIL-11 alone could shorten the recovery time compared with rhIL-11 combined with Plt transfusion [(8.0±2.6) d vs. (9.6±3.5) d, t=2.17, P=0.03]. Conclusion rhIL-11 twice a day of subcutaneous injection can effectively promote Plt recovery and reduce Plt transfusion with less adverse reactions, which is worthy of further application.

Objective To investigate the effects of simulated microgravity by rotary cell culture system (RCCS) on expression profiles of long non-coding RNA (lncRNA) in mouse fibroblasts L929 cell line.Methods L929 cells were cultured in vitro and randomly divided into simulated microgravity (SMG) group and normal gravity (NG) group.Each group had three samples,the rotator axis of SMG group was paralleled to the ground rotation,while the rotator axis of NG group was vertical to the ground rotation,and the speed of rotation was consistent for the two groups.The samples of two groups were collected on 7th day of culture and the total RNAs were extracted,labeled and hybridized in sequence.The lncRNA and mRNA were detected by Agilent Mouse lncRNA Chips respectively.Differentially expressed lncRNA were identified and then validated by RT-qPCR.GO and Pathway analysis were applied to determine the functional distribution of these target genes.The integration predictions of the lncRNA and mRNA co-expression had been proposed to refine the functional lncRNA-mRNA relationships.Results There were 238 differentially expressed lncRNAs including 134 lncRNAs up-regulated and 104 lncRNAs down-regulated,and 237 differentially expressed mRNAs including 53 mRNAs up-regulated and 184 mRNAs down-regulated significantly in mouse fibroblasts L929 cell line under simulated microgravity by RCCS.The RT-qPCR showed a high concordance with chip microarray results in 4 differentially expressed lncRNA.GO analysis showed that the differentially expressed lncRNAs were related to the biological processes such as negative regulation of megakaryocyte differentiation and negative regulation of wound healing.Pathway analysis showed that these target genes were related to the signal pathways of systemic lupus erythematosus and TGF-β.The lncRNA-mRNA co-expression networks were also established.Conclusion The simulated microgravity by RCCS could significantly affect the expression profiles of lncRNA and mRNA in mouse fibroblasts L929.The lncRNA target genes prediction and functional enrichment analysis based on gene chip technology may provide the theoretical basis for illustrating the mechanism and management of weightlessness stress injury.

Objective To analyze the A TP7A gene mutations in 2 related Chinese patients with Menkes disease (MD) and other members of the family and their hereditary features. Methods Two patients were clinically diagnosed as having MD. All 23 exons and exon-intron boundaries of ATP7A gene were polymerase chain reaction (PCR)-amplified and directly sequenced for genomic DNA extracted from the peripheral blood of both 2 patients and other members of the family; healthy controls were employed too. The mutations were proved by fluorescence quantitative PCR. Results Gross deletions from exon 2-12 were found in these 2 patients,respectively; their mothers,grandmother and aunt with normal phenotype carried those heterozygous mutations in the same site of A TP7A gene.Conclusions The 2 patients with MD are identified by gene and gross deletions from exon 2-12 are reported.