AMP-activated protein kinase (AMPK) is a widely distributed and evolutionarily conserved serine/threonine protein kinase present in eukaryotic cells. In regulating cellular energy metabolism, AMPK plays an extremely important role as an energy metabolic kinase. When the body is in a low energy state, AMPK is activated in response to changes in intracellular adenine nucleotide levels and is bound to adenosine monophosphate (AMP) or adenosine diphosphate (ADP). Activated AMPK regulates various metabolic processes, including lipid and glucose metabolism and cellular autophagy. AMPK directly promotes autophagy by phosphorylating autophagy-related proteins in the mammalian target of rapamycin complex 1 (mTORC1), serine/threonine protein kinase-dysregulated 51-like kinase 1 (ULK1) and type Ⅲ phosphatidylinositol 3-kinase-vacuolar protein-sorting 34 (PIK3C3-VPS34) complexes. AMPK also indirectly promotes autophagy by regulating the expression of downstream autophagy-related genes of transcription factors such as forkhead box O3 (FOXO3), lysosomal function transcription factor EB (TFEB) and bromodomain protein 4 (BRD4). AMPK also regulates mitochondrial autophagy, induces the division of damaged mitochondria and promotes the transfer of the autophagic response to damaged mitochondria. Another function of AMPK is to regulate mitochondrial health by stimulating mitochondrial biogenesis and participating in various aspects of mitochondrial homeostasis regulation. This review discusses the specific regulation of mitochondrial biology and internal environmental homeostasis by AMPK signaling channels as central to the cellular response to energy stress and regulation of mitochondria, highlighting the key role of AMPK in regulating cellular autophagy and mitochondrial autophagy, as well as advances in research on the regulation of mitochondrial homeostasis.

Objective To evaluate the clinical efficacy and clearance rate of pathogens of meropenem by different infusion therapies in treatment of severe sepsis for pediatric patients.Methods Sixty inpatients with serious sepsis from Pediatric Intensive Care Unit,Women and Children's Hospital of Ningbo University were randomly divided into three groups 20 in each.The meropenem was given according to the recommended therapeutic dose.Group A:The micropump was pumped for 0.5h at a rate of 100ml/h.Group B:The micropump was pumped for 3h at a rate of 16.7ml/h.Group C:Half of the menopenen was pumped for 0.5h at a rate of 50ml/h,and the other half was pumped for 2.5h at a rate of 10ml/h.Changes of indexes were compared before and after the treatment to estimate of the clinical effective rate,clearance rate of pathogens and side effects among the groups.Results There were observable improvements in the inflammatory indicators after treatment,such as white cell count,C-reactive protein and procalcitonin,etc.The total effective rate was 60％in group A,75％in group B,and 95％in group C.There were statistically differences among the groups(P＜0.05).Meanwhile,the bacterial clearance rates of the groups were 50％(group A),70％(group B)and 100％(group C),respectively(P＜0.05).The rate of side effect was 20％(group A),15％(group B)and 20％(group C),respectively(P＞0.05).Conclusion It can significantly improve the clinical efficacy and antibacterial efficacy by the two step infusion therapy of meropenem,compared with traditional infusion therapy and extended infusion.

OBJECTIVE: To investigate whether the Runx2 gene can induce the differentiation of human amniotic mesenchymal stem cells (hAMSCs) to ligament fibroblasts in vitro and promote the tendon-bone healing in rabbits. METHODS: hAMSCs were isolated from the placentas voluntarily donated from healthy parturients and passaged, and then identified by flow cytometric identification. Adenoviral vectors carrying Runx2 gene (Ad-Runx2) and empty vector adenovirus (Ad-NC) were constructed and viral titer assay; then, the 3rd generation hAMSCs were transfected with Ad-Runx2 (Ad-Runx2 group) or Ad-NC (Ad-NC group). The real-time fluorescence quantitative PCR and Western blot were used to detect Runx2 gene and protein expression to verify the effectiveness of Ad-Runx2 transfection of hAMSCs; and at 3 and 7 days after transfection, real-time fluorescence quantitative PCR was further used to detect the expressions of ligament fibroblast-related genes [vascular endothelial growth factor (VEGF), collagen type Ⅰ, Fibronectin, and Tenascin-C]. The hAMSCs were used as a blank control group. The hAMSCs, hAMSCs transfected with Ad-NC, and hAMSCs were mixed with Matrigel according to the ratio of 1 : 1 and 1 : 2 to construct the cell-scaffold compound. Cell proliferation was detected by cell counting kit 8 (CCK-8) assay, and the corresponding cell-scaffold compound with better proliferation were taken for subsequent animal experiments. Twelve New Zealand white rabbits were randomly divided into 4 groups of sham operation group (Sham group), anterior cruciate ligament reconstruction group (ACLR group), anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-NC-scaffold compound group (Ad-NC group), and anterior cruciate ligament reconstruction+hAMSCs transfected with Ad-Runx2-scaffold compound group (Ad-Runx2 group), with 3 rabbits in each group. After preparing the ACL reconstruction model, the Ad-NC group and the Ad-Runx2 group injected the optimal hAMSCs-Matrigel compunds into the bone channel correspondingly. The samples were taken for gross, histological (HE staining and sirius red staining), and immunofluorescence staining observation at 1 month after operation to evaluate the inflammatory cell infiltration as well as collagen and Tenascin-C content in the ligament tissues. RESULTS: Flow cytometric identification of the isolated cells conformed to the phenotypic characteristics of MSCs. The Runx2 gene was successfully transfected into hAMSCs. Compared with the Ad-NC group, the relative expressions of VEGF and collagen type Ⅰ genes in the Ad-Runx2 group significantly increased at 3 and 7 days after transfection ( P<0.05), Fibronectin significantly increased at 3 days ( P<0.05), and Tenascin-C significantly increased at 3 days and decreased at 7 days ( P<0.05). CCK-8 detection showed that there was no significant difference ( P>0.05) in the cell proliferation between groups and between different time points after mixed culture of two ratios. So the cell-scaffold compound constructed in the ratio of 1∶1 was selected for subsequent experiments. Animal experiments showed that at 1 month after operation, the continuity of the grafted tendon was complete in all groups; HE staining showed that the tissue repair in the Ad-Runx2 group was better and there were fewer inflammatory cells when compared with the ACLR group and the Ad-NC group; sirius red staining and immunofluorescence staining showed that the Ad-Runx2 group had more collagen typeⅠ and Ⅲ fibers, tending to form a normal ACL structure. However, the fluorescence intensity of Tenascin-C protein was weakening when compared to the ACLR and Ad-NC groups. CONCLUSION Runx2 gene transfection of hAMSCs induces directed differentiation to ligament fibroblasts and promotes tendon-bone healing in reconstructed anterior cruciate ligament in rabbits.
Pregnancy ; Female ; Humans ; Rabbits ; Animals ; Vascular Endothelial Growth Factor A/metabolism* ; Fibronectins/metabolism* ; Collagen Type I/genetics* ; Tenascin/metabolism* ; Collagen/metabolism* ; Anterior Cruciate Ligament/surgery* ; Mesenchymal Stem Cells ; Tendons/metabolism* ; Fibroblasts/metabolism*

Objective:To explore whether the lipopolysaccharide (LPS)-induced modification of O-linked N-acetylglucosamine (O-GlcNAc) is involved in the inflammatory signaling pathway of endothelial cells.Methods:Human umbilical vein endothelial cells (HUVEC) were cultured in vitro, and cells in logarithmic growth phase were used for experiments. Cells were divided into blank control group, LPS group (2 000 mg/L LPS), O-GlcNAc transferase (OGT) overexpression (OGT-OE)+LPS group (plasmid transfection OGT+2 000 mg/L LPS), protein kinase C (PKC) inhibitor+LPS group (10 μmol/L Go 6983+2 000 mg/L LPS), RhoA inhibitor+LPS group (40 μmol/L Rhoin hydrochloride+2 000 mg/L LPS), phosphatidylinositol-3-kinase (PI3K) inhibitor+LPS group (1 μmol/L SL-2052+2 000 mg/L LPS), serine/threonine kinase (Akt) inhibitor+LPS group (10 μmol/L PP2+2 000 mg/L LPS) and small interfering RNA (siRNA) treated Akt (si-AKT)+LPS group (si-Akt+2 000 mg/L LPS). After 24 hours of LPS treatment, real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the transcription levels of inflammatory cytokines [interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1)]. The protein expression or phosphorylation of OGT, O-GlcNAc, Akt, extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), nuclear factor-κB p65 (NF-κB p65), and signal transducer and activator of transcription 3 (STAT3) were determined by Western blotting. Results:Compared with the blank control group, the expression of OGT and the modification of O-GlcNAc in the LPS group were decreased, while the expressions of phosphorylated ERK, p38MAPK, and STAT3 were increased, and the transcript levels of inflammatory cytokines were also significantly increased [IL-6 mRNA (2 -ΔΔCt): 4.71±0.60 vs. 1.03±0.29, TNF-α mRNA (2 -ΔΔCt): 1.89±0.11 vs. 1.04±0.35, ICAM-1 mRNA (2 -ΔΔCt): 2.06±0.18 vs. 1.02±0.21, VCAM-1 mRNA (2 -ΔΔCt): 2.94±0.57 vs. 1.01±0.17, all P < 0.05], indicating that LPS could decrease O-GlcNAc modification, activate inflammatory signaling pathways and increase inflammatory cytokines expression. Compared with the LPS group, the expressions of phosphorylated ERK, p38MAPK, NF-κB p65, and STAT3 in the endothelial cells of the OGT-OE+LPS group were decreased, and the expression of inflammatory factors were significantly decreased [IL-6 mRNA (2 -ΔΔCt): 0.12±0.01 vs. 0.90±0.17, TNF-α mRNA (2 -ΔΔCt): 0.31±0.01 vs. 0.91±0.14, ICAM-1 mRNA (2 -ΔΔCt): 0.64±0.02 vs. 1.13±0.16, VCAM-1 mRNA (2 -ΔΔCt): 0.11±0.01 vs. 0.93±0.11, all P < 0.05], indicating that the increase of OGT level could inhibit the partial activation of the endothelial inflammatory signal pathway under the LPS stimulation. Compared with the blank control group, the phosphorylation level of Akt in the LPS group was increased. Compared with the LPS group, both OGT expression and O-GlcNAc modification were down-regulated after pretreatment of PKC inhibitor, RhoA inhibitor, PI3K inhibitor, or Akt inhibitor. Compared with the LPS group, the transcript levels of IL-6, TNF-α and ICAM-1 in the PP2+LPS group were significantly decreased [IL-6 mRNA (2 -ΔΔCt): 1.46±0.16 vs. 3.55±0.87, TNF-α mRNA (2 -ΔΔCt): 0.98±0.14 vs. 1.76±0.10, ICAM-1 mRNA (2 -ΔΔCt): 1.39±0.24 vs. 2.04±0.13, all P < 0.05], but there was no significant change in VCAM-1. Compared with the LPS group, the expression of OGT and O-GlcNAc modification in the si-Akt+LPS group were decreased, while the transcript levels of inflammatory cytokines were also significantly decreased [IL-6 mRNA (2 -ΔΔCt): 0.75±0.03 vs. 0.99±0.09, TNF-α mRNA (2 -ΔΔCt): 0.69±0.01 vs. 1.10±0.08, ICAM-1 mRNA (2 -ΔΔCt): 0.76±0.01 vs. 0.99±0.02, VCAM-1 mRNA (2 -ΔΔCt): 0.93±0.08 vs. 1.20±0.21, all P < 0.05], indicating that Akt participated in the action process of LPS on OGT and affected the inflammatory factor expression. Conclusions:The decreased level of O-GlcNAc modification in endothelial cells stimulated with LPS promotes partial activation of inflammatory signaling pathways, mainly involving ERK, p38MAPK, and STAT3, and affects the expression of inflammatory factors. AKT may be involved in the effect of LPS on the inhibition of O-GlcNAc modification.

Objective:To investigate pediatric sepsis-related mortality of pediatric intensive care unit(PICU) and family socioeconomic status in Yangtze River Delta.Methods:A prospective, multicenter observational study was conducted to collect sepsis cases from eight PICUs in Jiangsu, Zhejiang and Shanghai from August 2016 to July 2017.Sepsis cases were divided into normal sepsis group and severe sepsis group.The primary outcome was in-hospital death.Patient data were prospectively collected including age, gender, medical insurance status, long-term residence, source of admission, first-day pediatric sequential organ failure score(pSOFA) score, underlying diseases and socioeconomic characteristics including family education level, family annual economic income.Results:A total of 4, 983 patients admitted in PICUs, of which 651 patients were diagnosed sepsis on admission.The prevalence of sepsis was 13.1% (651/4 983), and overall mortality was 11.7% (76/651). The prevalence of severe sepsis was 28.3% (184/651), and the mortality was 20.1% (37/184). The overall median age was 0.9 years old.The infant group accounted for 50.8%, including 331 cases, followed by toddler group 19.8% (129 cases), preschool group 13.0% (86 cases), school group 11.8% (77 cases), and adolescent group 4.3% (28 cases). The median pSOFA score was 4.Logistic regression analysis showed that the OR value was 1.4(95% CI 1.3-1.5) of pSOFA score corresponding to the death of sepsis in hospital.There were 14.6% patients left hospital in medical insurance group, while 27.4% in non-medical insurance group, and there was significant difference between these two groups.The median of daily cost was 5, 446 RMB, among which the median of daily cost of sever sepsis was 6, 678 RMB.The median of total cost for sepsis was 36, 109 RMB, and that for severe sepsis was 41, 433 RMB. Conclusion:The sepsis-related mortality was high in PICU.The pSOFA score has a certain predictive value for the prognosis of sepsis.The burden of sepsis is still heavy.Compared with medical insurance families, non-medical insurance families have a higher proportion of choosing left hospital.

Objective To investigate the changes of plasma microRNA-223 (miR-223) in pediatric severe pneumonia patients and its relationship with severe pneumonia.Methods There were 50 children with severe pneumonia enrolled in the study (observation group),and 50 healthy children were selected as control group (normal control group).The expression levels of plasma miR-223 were detected by real time polymerase chain reaction.The relationship between miR-223 and procalcitonin (PCT),C-reactive protein (CRP),tumor necrosis factor-α (TNF)-α,and interleukin (IL)-10 were analyzed.The predictive value of miR-223 in plasma,PCT,and CRP to severe pneumonia was evaluated by receiver operating characteristic (ROC) curve.The relationship between miR-223,T cells,TNF-α,and IL-10 was analyzed.Results The plasma miR-223 expression levels in observation group were upregulated compared to those in the normal control group [(16.01 ± 5.17) × 10-4 mg/L vs (5.44 ± 2.21) × 10-4 mg/L,t =7.46,P ＜ 0.01].The CRP and PCT expression levels in severe pneumonia patients were higher than those of the normal control group [z =5.496,5.198,P ＜0.05,orP ＜0.01].The expressions of CD4+ CD25+Treg,miR-223,and IL-10 in observation group were higher than those of normal control group (P ＜0.05).The expression of CD4 + CD25 + Treg,miR-223,and IL-10 in the death children were higher.There was a positive correlation between miR-223 and IL-10 (r =0.335,0.571,P ＜ 0.01).The sensitivity and specific degrees to predict the severe pneumonia in mir-223,PCT,and CRP were 83.79％,86.12％,66.68％,91.05％,78.01％,and 44.23％,respectively.miR-223 was better than PCT and CRP to predict severe pneumonia in children.Conclusions The expression levels of plasma miR-223 in children with severe pneumonia could reflect the immunity,and it can be used as early prognostic markers to reflect the severity of inflammation in some degree.

Objective To observe the effect of acupuncture at nape points plus nerve electrical stimulation on deglutition function and cerebral blood flow in pseudobulbar palsy after cerebral stroke. Method The eligible subjects were randomized into a control group of 35 cases and a treatment group of 33 cases. The control group was intervened by conventional Western medication, while the treatment group was by acupuncture at nape points plus nerve electrical stimulation in addition to the Western medication, once a day, 4 weeks as a treatment course. Result The total effective rates were respectively 93.9%and 80.0%in the two groups, and the treatment group was significantly superior to the control group; after the intervention, the high whole blood viscosity, low whole blood viscosity, hematocrit, and plasma viscosity all dropped markedly in the treatment group, and were significantly different from that in the control group (P<0.05). The Transcranial Doppler (TCD) showed that the blood flow mean velocity (Vm), systolic velocity (Vs), and pulsatility index (PI) of the left vertebral artery, right vertebral artery, and basilar artery in the treatment group were significantly improved after the intervention (P<0.05), and the Vm and Vs of the left vertebral artery, right vertebral artery, and basilar artery in the control group were significantly improved after the intervention (P<0.05), while PI in the control group didn’t show significant improvement (P>0.05). After the intervention, there were significant differences in comparing the Vm, Vs, and PI of the left vertebral artery, right vertebral artery, and basilar artery between the two groups (P<0.05). Conclusion Acupuncture at nape points plus nerve electrical stimulation is effective in improving the deglutition function in pseudobulbar palsy after cerebral stroke, and its action mechanism is plausibly through improving the cerebral blood flow and promoting the recovery of brain function.

Objective To establish the data including anatomy and histology of main organs in Rongshui miniature pig (RMP).Methods F1 Rongshui miniature pigs with male and female (2 in each group) in 6 month old were used in this experiment.We measured body weights, dissected these pigs after anaesthesia, recorded total blood volume, total plasma volume, number of spine and dental formula, took main organs for photographs, and made histological sections observed and took photographs by microscope.Results We gained the photographs of main organs and histological sections, organ weights,organic coefficients and other basic data.Conclusion Basic anatomy and histology data of main organs in RMP were collected.

Objective To detect the blood parameter, blood biochemical and electrolyte indices of the F1 generation of Rongshui miniature pig ( RMP) .Methods The blood of 43 female and 42 male RMPs of 4 th month old, and 36 RMPs of 12th month old ( half male and female) were extracted from jugular vein.And the blood parameter, blood biochemical and electrolyte indices were detected by blood analyzer and automatic biochemical analyzer.Results In the same month-old RMP, no significant difference between male and female were found in most indices of blood parameter, blood biochemical and electrolyte indices.On the other hand, many indices were difference between 4th month old and 12th month old RMPs of same gender.Compared with the 4th month old RMP, the 12th month old RMP decreased significantly in WBC and PLT, increased in HGB ( P <0.05 ) while RBC was the same ( P >0.05 ) .Serum ALT, AST, ALP, CK (male), LDH(male), A/G, BUN, GLU (female), CHOL (male) and K+decreased significantly (P <0.05), while serum TP, TBIL, CR and Ca2+increased significantly (P <0.05),but serum CHOL, TG, HDL-C and LDL-C were not different.86.4%(19/22) biochemical and electrolyte indices in RMP were in/or close to the range of normal value of human.Conclusion Most of the blood parameter, blood biochemical and electrolyte indices of RMP were close to human’ s normal value.

The provenances of Rongshui miniature pigs ( RMPs) were purchased from Rongshui, Guangxi, in 2012.130 RMPs were transported to Sanshui, Guangdong,China, which were breed according to the laboratory animal standards.83 RMPs were selected randomly from the first filial generations ( F1 ).The basic data were collected including breed characteristics, reproductive performance, grow curve, hematology, biochemical markers of serum and urine, organ coefficient, Chromosome analysis.According to the national and local standards, the quality control standards of RMP were set up including microbiological, parasitic, environment and facilities, fodder, pathology, genetic.The results showed that RMPs adapt to the climate of Guangdong.The natural mating and conceive rate was 88.3% with the pregnancy of 112 days.The average number of firstborn and still-born was 6.1 and 7.9 respectively.RMP was small body size with the adult body weight of 17.21 ±5.20 kg and 16.35 ±5.23 kg in female and male respectively.RMP was very tame.The mitochondrial genome analysis suggested RMP belonged a typical miniature pig breed in China, which is ancient than Lanyu pig.RMP could be breed as a new kind of laboratory animal.