Background and objectives:Hepatic steatosis and inflammation are key characteristics of non-alcoholic fatty liver disease(NAFLD).However,whether and how hepatic steatosis and liver inflammation are differentially regulated remains to be elucidated.Considering that disruption of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3(Pfkfb3/iPfk2)dissociates fat deposition and inflammation,the present study examined a role for Pfkfb3/iPfk2 in hematopoietic cells in regulating hepatic steatosis and inflammation in mice. Methods:Pfkfb3-disrupted(Pfkfb3+-)mice and wild-type(WT)littermates were fed a high-fat diet(HFD)and examined for NAFLD phenotype.Also,bone marrow cells isolated from Pfkfb3+/-mice and WT mice were differentiated into macrophages for analysis of macrophage activation status and for bone marrow transplantation(BMT)to generate chimeric(WT/BMT-Pfkfb3+/-)mice in which Pfkfb3 was disrupted only in hematopoietic cells and control chimeric(WT/BMT-WT)mice.The latter were also fed an HFD and examined for NAFLD phenotype.In vitro,hepatocytes were co-cultured with bone marrow-derived macrophages and examined for hepatocyte fat deposition and proinflammatory responses.Results:After the feeding period,HFD-fed Pfkfb3+/-mice displayed increased severity of liver inflam-mation in the absence of hepatic steatosis compared with HFD-fed WT mice.When inflammatory activation was analyzed,Pfkfb3+/-macrophages revealed increased proinflammatory activation and decreased anti-proinflammatory activation.When NAFLD phenotype was analyzed in the chimeric mice,WT/BMT-Pfkfb3+/-mice displayed increases in the severity of HFD-induced hepatic steatosis and inflammation compared with WT/BMT-WT mice.At the cellular level,hepatocytes co-cultured with Pfkfb3+/-macrophages revealed increased fat deposition and proinflammatory responses compared with hepatocytes co-cultured with WT macrophages. Conclusions:Pfkfb3 disruption only in hematopoietic cells exacerbates HFD-induced hepatic steatosis and inflammation whereas the Pfkfb3/iPfk2 in nonhematopoietic cells appeared to be needed for HFD feeding to induce hepatic steatosis.As such,the Pfkfb3/iPfk2 plays a unique role in regulating NAFLD pathophysiology.

Aim To investigate the skeletal effects of ginseng flower bud(GF)on osteopenia induced by D-galactose using histomorphometry and biomechanical properties.Methods Fifty three-month-old male Sprague-Dawley rats were randomly divided into five groups.Rats in NS group(NS)were treated with NS(5 mL·kg-1·d-1)by subcutaneous injection and daily oral gavage with vehicle as control.Rats in the other four groups were given D-galactose at the dose of 100 mg·kg-1·d-1 by subcutaneous injection.Solvent control was performed between NS and DG: gastric irrigation with distilled water of 10 mL·kg-1·d-1.Other groups were: CP was gastric irrigated with integrated medicine(stanozolol 0.54 mg·kg-1·d-1+piracetam 432 mg·kg-1·d-1),GF(L)with ginseng flower bud of 0.486 g·kg-1·d-1 and GF(H)with ginseng flower bud of 2.43 g·kg-1·d-1 for 14 weeks.The longitudinal proximal tibial metaphyseal(PTM),the fifth lumbar vertebral body(LVB)and tibial shaft(Tx)sections were performed undecalcifiedly and used for bone histomorphometric analysis.858 Mini Bionix materials testing system was used to analyze the biomechanic properties of right femur via three-point bending test.The left femur was dried and assimilated,whose bone calcium(Ca),phosphate(P)content and bone hydroxyproline content were tested.Results Compared with D-glagatose group,in PTM of D-galactose treated rats,the％Tb.Ar was increased both in GF(L)and GF(H)treated groups.While the Tb.Sp was decreased.％Oc.S.Pm and Oc.N/mm decreased in GF(L),and those in GF(H)were decreased as well.In Tx,％Ct.Ar was raised,while％Ma.Ar was decreased in GF(L)and GF(H).The elastic load of femur was increased.Conclusions Compared with DG group,there are significant differences in bone histomorphometry of Tx and PTM in all doses of GF,but no significant changes are detected in hydroxyproline,Ca,and P content of femur.

Aim To investigate the effects of predni-sone on trabecular microstructure and biomechanical properties of femur in a rat model of type II collagen-induced arthritis (CIA ) using micro-CT and biome-chanics.Methods Forty 8-week-old male Lewis rats were randomly divided into 2 groups:control (CON ) group with 6 rats,and the remaining 34 rats were used to establish the CIA model.3 weeks after immunization screening CIA rats were randomly divided into CIA group,CIA plus prednisone 4.5 mg · kg-1 · d -1 group and CIA plus prednisone 9 mg · kg-1 · d -1 group.Rats in CON group were given vehicle as well as in CIA group.Rats in the other two groups were treated with prednisone at 4.5 mg·kg-1 ·d -1 or 9 mg ·kg-1 · d -1 .After 90 days treatment,all rats were euthanized,and the left femur was collected for biome-chanics,micro-CT scanning and three-dimensional re-construction.Results Micro-CT data showed that tra-becular thickness,trabecular number,bone volume/total volume,bone mineral density in CIA group were significantly lower than those in CON group.While tra-becular separation,structure model index were signifi-cantly higher than those in CON group.Compared with CON group,biomechanical properties (elastic load, maximum load,break load and stiffness)were signifi-cantly decreased in CIA group.Compared with CIA group,bone volume/total volume and trabecular num-ber were increased,while trabecular separation was significantly decreased in two prednisone groups.Com-pared with CIA group,there was no significant change in biomechanical properties in two prednisone groups. Conclusions Treatment with prednisone for 3 months can ameliorate the damage of trabecular microstructure of the femur in CIA rats,but it has no effect on biome-chanical properties and bone mineral density.

Objective Anatomical researches of two long superiror genicular vessels,the descending branch of lateral circumflex femoral artery (LCFA) and the descending genicular artery(DGA),were performed to discuss the feasibility of reconstruction in lower leg's destruction injury by free flaps transplant anastomoses with these two long superior genicular vessels.Methods Thirty embalmed lower limb specimens from adult cadavers perfused with red latex were used for this anatomical study.The superior of patella,anterior inferior iliac spine and adductor tubercle were used as observation landmark.Dissection started along the line that from the middle point of Inguinal Ligament to the middle point of superior line of patella,and dividedly turned over to posterior lateral part and posterior medial part.The followings were focused observed:①The external diameter at the beginning of the D-LCFA ; the 0.5 mm external diameter point of this artery,and its length to the beginning and superior of patella.②The external diameter at the beginning of DGA,the distance between the beginning of DGA and adductor tubercle.③Anastomoses relations of final branches of D-LCFA and DGA with other arteries around the knee.Results The external diameter of the beginning of D-LCFA was (2.73 ± 0.35) mm ; the 0.5 mm external diameter point of this artery's length to the beginning and to the superior of patella were (24.56 ± 0.92) cm and (6.09 ± 0.86) cm; both D-LCFA and DGA had sent out many perforator arteries on their ways; the final branch of D-LCFA and DGA had lots of anastomoses relations in perioseal deep fascia and superficial fascia with other genicular arteries.Conclusion Base on the anatomical researches,in theory,the two long superior genicular vessels (D-LCFA and DGA) have enough lengthes and blood supply to regress and anastomosis with free flaps transplant to repair lower leg's smashed wound.

Artemisinin-based combination therapies (ACTs) are recommended to be the most effective therapies for the first-line treatment of uncomplicated falciparum malaria. However, artemisinin is often in short supply and unaffordable to most malaria patients, which limits the wide use of ACTs. Production of amorpha-4,11-diene, an artemisinin precursor, was investigated by engineering a heterologous isoprenoid biosynthetic pathway in Escherichia coli. The production of amorpha-4,11-diene was achieved by expression of a synthetic amorpha-4,11-diene synthase gene in Escherichia coli DHGT7 and further improved by about 13.3 fold through introducing the mevalonate pathway from Enterococcus faecalis. After eliminating three pathway bottlenecks including amorpha-4,11-diene synthase, HMG-CoA reducase and mevalonate kinase by optimizing the metabolic flux, the yield of amorpha-4,11-diene was increased by nearly 7.2 fold and reached at 235 mg/L in shaking flask culture. In conclusion, an engineered Escherichia coli was constructed for high-level production of amorpha-4,11-diene.
Alkyl and Aryl Transferases ; genetics ; Antimalarials ; metabolism ; Artemisinins ; metabolism ; Enterococcus faecalis ; genetics ; Escherichia coli ; genetics ; metabolism ; Metabolic Engineering ; methods ; Phosphotransferases (Alcohol Group Acceptor) ; metabolism ; Sesquiterpenes ; metabolism ; Transformation, Bacterial

Objective To construct human apolipoprotein O (apolipoprotein O, ApoO) expression vector and obtain recombinant fusion protein thioredoxin (Trx)-ApoO by pET prokaryotic expression system. Methods The ApoO gene fragment from the human liver cDNA library was amplified by PCR. The resulting product was cloned into pET-32a(+) vector and sequenced. The confirmed cDNA was cloned into plasmid E.coli DH10B and then transformed into E.coli BL 21 (DE3) where it was induced to express protein by isopropyl β-D-1-thiogalactopyranoside (IPTG).The fusion protein was purified by Ni-NTA resin. Results The ApoO gene was cloned by PCR and a 519 bp DNA fragment was shown on the agarose electrophoresis. The cloned gene was sequenced and demonstrated to have the same sequence as that of human ApoO gene in GenBank which justified a successful construction of recombinant plasmid. ApoO cDNA gene fragment was induced by IPTG, and a 34 kD recombinant fusion protein Trx-ApoO was tested on sodium dodecyl sulfate polyacrylamide (SDS-PAGE). Conclusion Human ApoO gene is successfully cloned and its recombinant fusion protein Trx-ApoO is expressed.

This investigation was directed to the effects of DanShenGuBao on biomechanical properties and bone mineral density (BMD) of femur induced by retinoic acid (RA) in rats. Forty 4-month-old virgin female Sprague-Dawley rats were randomly divided into 5 groups(8 rats each) control group, RA group and different doses of DanShenGuBao groups. Rats in control group were given vehicle, rats in other four groups were given RA at 70 mg x kg(-1) x d(-1) in the morning and given different drugs in the afternoon at the same time. Rats in RA group were given vehicle, rats in other groups were given different doses of DanShenGuBao which contained 10 mg x kg(-1) x d(-1), 5 mg x kg(-1) x d(-1), 2.5 mg x kg(-1) x d(-1) tanshinol, respectively. All of rats were treated at 5 ml x kg(-1) by oral gavaged for 28 days. In preparation for the determination of dynamic changes in bone tissues, all rats were given subcutaneous injections of 30 mg x kg(-1) tetracycline on the 14th, 13th day and 5 mg x kg(-1) calcein on the 4th, 3rd day before death. At the experimental endpoint, the rats were sacrificed by cardiac puncture under sodium pentobarbital anesthesia. Physical parameters, BMD and biomechanical properties of femur were assessed. Compared with those in control group, the physical parameters (cross-sectional diameter, wet and dry weight), BMD and biomechanical properties (max-load, elasticity-load, break-strain, rigid coefficient and bending-energe) were significantly decreased in RA group. Compared with that in RA group, the BMD of femur was increased significantly in medium and high dose of DanShenGuBao group, but there was no significant change in physical parameters and biomechanical properties of femur. RA could decrease the physical parameters, BMD and biomechanical properties of femur. DanShenGuBao could increase BMD, but it was found with no obvious effect on physical parameters and biomechanical properties.
Absorptiometry, Photon ; Animals ; Biomechanical Phenomena ; drug effects ; Bone Density ; drug effects ; Drugs, Chinese Herbal ; therapeutic use ; Female ; Femur ; diagnostic imaging ; drug effects ; Osteoporosis ; chemically induced ; drug therapy ; Phytotherapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza ; chemistry ; Tretinoin

3T3-L1 adipocytes were cultured with various concentrations of high-density lipoprotein ( HDL, 0, 10, 50, and 100 μg/ml ) for 16 h and with lipopolysaccharide ( LPS, 100 ng/ml ) for another 6 h. Interleukin-8 in the medium was determined by ELISA, and PPAR-γ mRNA expression by reverse transacription polymerase chain reaction (RT-PCR). Interleukin-8 levels were increased in LPS-treated cells ( P＜0.05 ), but decreased in HDL-treated cells in the dose-dependent manner. PPARγ mRNA expressions were increased in HDL-treated groups than those treated only with LPS. These results suggested HDL may decrease interleukin-8 secretion via up-regulating PPARγ expression in adipocytes.

Aim To investigate the effects of retinoic acid (RA) on induction osteroporosis model rats andpreventive effects of Danshengubao.Methods 4-month-old female Sprague-Dawley rats were given RA at 70 mg·kg~(-1)·d~(-1) and were given Danshengubao at different doses at the same time.All rats were treated by oral gavaged for 28 days.The static and dynamic parameters in cancellous bone of the fifth lumbar vertebrae (LV5) were examined and the dynamic changes of the tibial shaft (Tx) were observed with histomorphometrical analyses; the forth lumbar vertebrae (LV4) was used to perform the compression test.Results Compared with control group, biomechanical properties of LV4, the static parameters ( total tissue area, trabecular area, trabecular perimeter) and the dynamic parameters of LV5 were significantly decreased in RA group.Compared with control group, bone formation of Tx was decreased in periosteal surfaces but enhanced in endocortical surfaces in RA group.Compared with RA group, the biomechanical properties of LV4 were increased significantly in low and medium dose of Danshengubao groups.Conclusion sRA can decrease the size and the biomechanical properties of LV, but it can not change the percentage trabecular area. The mechanism may be related to the act that RA can inhibit cancellous bone formation, decrease the modeling of cortical bone in periosteal surfaces and enhance the remolding of cortical bone in endocortical surfaces. Danshengubao can improve biomechanics of LV induced by RA in rats.

BACKGROUND: Prevention of male osteoporosis is attracting more and more attention. OBJECTIVE: To study the effect of etidronate disodium on different skeletal sites by using bone histomorphometry through establishing a castrated rat model of osteoporosis. DESIGN, TIME AND SETTING: A completely randomized grouping controlled animal experiment was performed in the Animal Experimental Center and Department of Bone Biology Laboratory, Guangdong Medical College between October 2002 and September 2006. MATERIALS: Forty 3.5-month-old male Sprague Dawley rats, weighing (299+_22) g, were selected. Etidronate disodium was produced by Chengdu Chemical Pharmaceutical Factory with the batch number of 970101. Methyltestosterone was produced by Guangzhou Pharmaceutical Jacob with the batch number of 990701. METHODS: Rats were randomly divided into five groups: control group, sham operation group, castration group, castration with methyltestosterone group and castration with etidronate disodium group, eight rats in each group. Rats in the control group were sacrificed at the beginning of the study. Rats in the sham operation group underwent skin incision to expose the testis, but not removed. The remaining rats were treated to remove the testis by the method reported in the literature. Rats in the sham operation group and castration group were given normal saline, rats in the castration with methyltestosterone group were given methyltestosterone at 1.8mg/kg/d, rats in the castration with etidronate disodium group were given etidronate disodium at 36mg/kg/d. All of the rats were treated with intragastric administration at 5mL/kg for 90 days. MAIN OUTCOME MEASURES: Bone histomorphometric analysis of the proximal tibial metaphysis (PTM), tibial shaft (Tx) and the fifth lumbar vertebral body (LVB) were performed in undecalcified sections. RESULTS: Compared with the sham operation group, trabecular area percentage (%Tb.Ar), trabecular number (Tb.N) or trabecular thickness (Tb.Th) of PTM and LVB in the castration group were decreased (P < 0.05 and 0.01 ), while trabecular separation (Tb.Sp), percent labeled perimeter (%L.Pm), bone formation rate (BFR/BV) and osteoclast number per mm (Oc.N) were increased (P < 0.05 and 0.01 ). Tb.Ar of PTM and LVB were increased both in the etidronate disodium group and in the methyltestosterone group compared to those of the castration group, while bone formation indices and bone resorption perimeter were decreased. There was no significant difference between the etidronate disodium and methyltestosterone groups, and no significant change was in Tx in all groups. CONCLUSION: Etidronate disodium can prevent the cancellous bone loss of PTM and LVB in castrated rats, but has no effects on the cortical bone of Tx.