AIM To investigate whether pioglitazone can protect cortical neurons from lipopolysaccharides(LPS)-induced neurotoxicity and the mechanisms responsible for this protective effect. METHODS After 7 d cultures，cultured cortical neurons were incubated with LPS 10 mg·L~(-1) for 4-24 h with or without other drugs. In co-incubation experiments, other drugs were added to the neurons 30 min or 1 h prior to incubation with LPS. The cell viability was assessed by MTT assay. The neuronal apoptosis was quantified by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst 33258 staining. The cultured cells were then fixed on the 7th day and immunocytochemically stained with phosphorylated JNK1 antibody. The protein expressions of active caspase 3 and phosphorylated JNK1 were measured by Western blot. Nitric oxide (NO) generation was measured by Griess method. RESULTS The decrease of cell viability and the increase of apoptotic cells in cultured cortical neurons were observed incubated with LPS for 24 h compared with the normal controls. The cell viability of cortical neurons was decreased from (100.0±10.9)% in the normal control group to (72.3±2.1)% in the LPS-treated group and the apoptotic cell percentages were increased from (11.5±4.2)% in the normal control group to (39.5±8.2)% in the LPS group. LPS induced the increases in phospho-JNK1, active caspase 3 expression, and NO generation. Pioglitazone 0.01, 0.1 and 1 μmol·L~(-1), respectively inhibited LPS-induced decrease in cell viability and increase of apoptotic morphology, active caspase 3 expression in cultured neurons. In LPS+pioglitazone 1 μmol·L~(-1) group, cell viability was （97.8±9.7）%, the apoptotic cells percentage was (20.6±5.0)%, NO generation (6.8±1.3)μmol·L~(-1). Furthermore, pioglitazone also inhibited LPS-induced the increase in JNK1 phosphorylation and NO generation. JNK inhibitor SP600125 5 μmol·L~(-1) significantly inhibited LPS-induced neurotoxicity, cell viability was increased from (72.3±2.1)% to (109.8±11.8)%, the apoptotic cells percentage from (39.5±8.2)% decreased to (19.1±4.8)%, NO generation from （21.1±5.0）μmol·L~(-1) decreased to（4.0±1.3）μmol·L~(-1). The PPARγ antagonist GW9662 10 μmol·L~(-1) did not reverse the effects of pioglitazone. In LPS+pioglitazone 1 μmol·L~(-1)+GW9662 10 μmol·L~(-1) group, cell viability was (90.7±6.9)%, the apoptotic cells percentage was (23.4±4.1)%, and NO concentration was (5.8±0.7)μmol·L~(-1). CONCLUSION Pioglitazone protects cortical neurons against LPS insult at least via inhibiting JNK activity and NO generation, but not PPARγ activation.

Objective To study the effects of using jaw tracking technique with Smart LMC algorithm on the absorbing dose of planning target volume (PTV) and organs at risk (OARs) in dynamic intensity-modulated radiation therapy (IMRT) for nasopharyngeal carcinoma (NPC).Methods Field fluencies of 10 cases of NPC patients were optimized using DVO algorithm on Eclipse TPS (11.0),and according to the same optimal fluence,MLC operation files were calculated using jaw tracking technique and jaw fixing technique respectively,dose distribution was calculated with AAA algorithm and jaw tracking IMRT plan (JT-IMRT) and jaw fixing IMRT plan (JF-IMRT) were generated respectively.Collimators' position at the plan implementation was observed,and the total number of plans' monitor units (MU),the dose of PTV,the absorb dose of OARs,and the actual fluence verification pass rate were compared.Results The collimators' opening gap distances in 166 control points of the JT-IMRT reduced in both X and Y directions in the field,compared to that of the JF-IMRT.Total number of the JT-IMRT's MU increased by 3.59％-11.63％.There was no statistical significant difference between the doses of the PTV.Statistical significance was found in the differences between maximum dose (Dmax) of brainstem,spinal cord,crystal,optic nerve,the mean dose (Dmean) and D50％ of parotid and their decreased values after therapy (t=5.70-8.66,P＜0.05).The actual fluence verification pass rate of the JT-IMRT was higher than that of the JF-IMRT.There was a significant difference between the results (t=5.18,P＜0.05).Conclusions The JT-IMRT plan of the smart LMC algorithm is more tolerant to the radiation leakage between inter-and intra-leaf.The dose of OARs is lower,while the dose calculation precision and the verification pass rate are higher,the actual radiation dose is more accurate and reliable.Therefore it is more suitable for clinical applications.

Background Stickler syndrome is a genetic connective tissue disorder that affects the ocular,skeletal,orofacial and auditory systems.To determine the gene mutation loci can offer a basis for genetic diagnosis and management of Stickler syndrome.Objective The aim of this study was to research the clinical characteristics of a pedigree with Stickler syndrome and identify the disease-causing gene mutation.Methods This study was approved by Ethic Committee of Peking Union Medical College Hospital.The clinical study and pedigree analysis were performed in one family with Stickler syndrome type Ⅰ (STL Ⅰ).Nine family members were examined with informed consent.The entire coding regions of COL2A1 gene with flanking intronic regions were amplified by PCR and directly sequenced.The detected sequence change was confirmed to be mutationloci by examining whether they existed in normal control individuals.Mutant proteins were predicted with online software.Results There were 4 generations and 11 members in this family,and 2 members died,including 1 patient.Three patients were found in 9living families.Inheritance of this family complicd with an autosomal dominant inheritance mode.All affected individuals showed the consistent phenotypes with STL Ⅰ,including high myopia,membranous vitreous anomaly and surface central flat,short nose,palatoschisis,etc.Mutation screening of COL2A1 gene revealed that the first base of intron 12 was deleted(IVS12+1G del).Nucleotide sequence analysis showed that this mutation led to the functional abnormal of this gene by forming termination cordon in advance.This mutation occurred in all affected individuals,however,no mutation was observed in any unaffected member or 100 normal unrelated individuals.Conclusions This study identifies a novel splice-site mutation(IVS12+ 1G del)in COL2A1 gene in a Chinese STL Ⅰ pedigree.This is the first report on a mutation in a Chinese STL Ⅰ family.

Aim To study the anti-tumor effect of 20(S)-Protopanaxadiol(PPD)at different concentration on human liver cell line SMMC-7721 in vivo and in vitro.Methods The subcutaneous transplantable tumor model of human liver cancer in nude mice was established and the anti-tumor effect was calculated.Cell growth rate was determined with MTT assay.The apoptosis was analyzed by FITC-AnnexinⅤ/PI and Hoechst33342 staining method,and the activity of Caspase-3 was detected.Results In vivo,PPD could obviously inhibit the growth of transplanted tumor.In vitro,PPD induced inhibition of human liver cancer SMMC-7721 cells was time-dependent and dose-dependent.The apoptotic body was observed by Hoechst33342 staining.PPD could induce cell apoptosis of SMMC-7721,and the increase of Caspase-3 activity was observed in each PPD group.Conclusion PPD could inhibit the growth of human liver cancer SMMC-7721 cells in vivo and in vitro by up-regulating the activity of Caspase-3 and inducing the cell apoptosis.

Objective To observe the effects of 860 MHz microwave radiation on an established dual task model of trace and delay eyeblink conditioning in guinea pigs.Methods Twenty-four guinea pigs with an established dual task model of trace and delay eyeblink conditioning were assigned randomly into four groups : a microwave-exposed 1 h group, a microwave-exposed 20 min group, a sham-exposed group and a normal control group.The guinea pigs in the 1 h and 20 min groups were irradiated on the head daily for 3 days using 860 MHz electromagnetic radiation at a power density of 1 mW/cm2 for 1 h and for 20 min, respectively. After radiation the guinea pigs were trained with classical dual task eyeblink conditioning.Results Compared with the normal control group, the behavioral parameters (acquisition rate and peak amplitude of trace and delay eyeblink) of guinea pigs in the microwave-exposed 1 h group had decreased significantly with no obvious change in latency. The behavioral parameters of guinea pigs in the microwave-exposed 20 min group, the sham-exposed group and the normal control group showed no obvious change.Conclusions Microwave radiation at 860 MHz and 1.0 mW/cm2 for 1 h can cause changes in dual trace and delay eyeblink conditioning in guinea pigs and decrease learning and memory capacity.

OBJECTIVETo explore the protective mechanism of electroacupuncture (EA) at "Neiguan" (PC 6) on ischemic myocardial injury, and to explain the response patterns and characteristics of the specific effect of acupoints along meridians in sodium channel in the level of cardiac organ.

METHODSA total of 60 SPF male rats were randomly divided into a blank group, a model group, a non-acupoint group, a Neiguan group and a Lieque group, 12 cases in each one. Except the blank group, rats in the remaining group were treated with subcutaneous injection of isoprenaline to establish the model of myocardial ischemia. Rats in the Neiguan group, Lieque group and non- acupoint group were treated with EA, dilatational wave, with a frequency of 2 Hz/20 Hz. The intensity was 2-3 mA. The needles were retained for 20 min per time, once a day for consecutive 7 days. In the blank group and control group, the rats were grasped and fixed at the treating time each day. The western-blot method was used to test the expression of voltage-gated sodium channel alpha subunit (Nav 1.5), protein tyrosine kinase (PTKs) and protein tyrosine phosphatase (PTPs).

RESULTSThe expression of Nav 1.5 and PTKs in the model group was lower than that in the blank group (both P<0. 01); the expression in the Neiguan group and Lieque group was higher than that in the model group (all P < 0.01); the expression of Nav 1.5 and PTKs in the Neiguan group was higher than that in the Lieque group (both P < 0.01). The expression of PTPs in the model group and non-acupoint group was higher than that in the blank group (both P < 0.01); the expression of PTPs in the Neiguan group and Lieque group was significantly down-regulated, which was lower than the model group (both P < 0.01); the down-regulation in the Neiguan group was significantly different from that in the Lieque group (P < 0.05).

CONCLUSIONEA at "Neiguan" (PC 6), by down-regulating the expression of PTPs, up-regulating the expression of Nav 1.5 and PTKs, is likely to achieve the aim of regulation on sodium channel activity and calcium overload, further to improve myocardial ischemia, which provides experimental basis for the theory of the specific effect of acupoints along meridians.

Acupuncture Points ; Animals ; Disease Models, Animal ; Electroacupuncture ; Humans ; Male ; Myocardial Ischemia ; genetics ; metabolism ; therapy ; Myocardium ; metabolism ; Rats ; Rats, Sprague-Dawley ; Sodium Channels ; genetics ; metabolism

Objective To study the inhibitory effects of δ-opioid receptor activation in serumdeprivation induced apoptosis of human liver cells and the proposed protein kinase C(PKC)pathway mechanism.Methods MTT assay was used to detect the survival rate of human liver cells in vitro and Annexin V-FITC/PI double staining was used to detect the cell apoptosis rate.Flow cytometry was used to analyze cell cycle,RT PCR used to analyze the PKC mRNA and Western Blot analysis was used for detecting the protein expression of PKC and Caspase-3.Results After serum-deprivation for 48h of cultured human liver cells in vitro,significant liver cell apoptosis occurred.The apoptosis was suppressed by δ-opioid receptor activation,which manifested as a slower rate of apoptosis,decreased expression of Caspase-3and increased expression of PKC.After GF109203X was added,the inhibitory effects of DADLE decreased markedly.Conclusion Activation of δ-opioid receptor on the membrane of human liver cells has inhibitory effects on serum-deprivation induced apoptosis of liver cells.The underlying mechanism may be associated with PKC pathway activation.

Objective To study the giant invasive pituitary tumor neuroendoscopic operation indications,operation excision,risk aversion,and the operation skills.Methods The clinical data of 7 patients with giant invasive pituitary tumor among of endoscopic transsphenoidal surgery 61 cases of neurological patients with pituitary tumors were analyzed retrospectively.Results There were 1 case of total resection,6 cases of subtotal resection invading cavernous sinus cases,diaphragma sellar was seen in 5 cases of resection of the tumor,and 2 cases showed no diaphragma sellar.The average operation time was 100 minutes.No intraoperative transfusion.Postoperative hemorrhage in 2 cases,and 1 death case in this group after 36 hours,and 1 case undergoing endoscopic hematoma resection and cured.Conclusions With the development of endoscopic techniques,indications for operation with the new changes,for the giant invasive pituitary tumor operation therapy,endoscopic technique provides a disposable operation resection,the method is safe and avoid catastrophic consequences.

Aim To investigate whether pioglitazone has protective effect against glutamate induced neurotoxicity in cultured cortical neurons and its possible molecular mechanisms underlying pioglitazone's neuroprotective effects.Methods The cortical neurons were taken from newborn rats and used for experiments 7 days after culture.The neurons were randomly divided into control group;glutamate group; glutamate+piogli-tazone group;glutamate+SP600125 group;SP600125 group.Cell viability was determined by MTT.The morphology change of neurons was observed under a fluorescence microscope with fluorescence dye Hoechst 33258.Immunostaining was used to investigate the expression of phospho-ATF2 in neuronal cells.Western blot was performed to investigate the protein level of phospho-JNK1 and total JNK1.Results Pioglitazone markedly reduced the damage of cortical neurons caused by glutamate.Pioglitazone also significantly inhibited glutamate induced up-regulation of phospho-JNK1 protein level and phospho-ATF2 expression.SP600125, an inhibitor of JNK, antagonized the toxicity induced by glutamate.Conclusions Pioglitazone can protect cultured cortical neurons from glutamate induced damage.The protective effect of pioglitazone appears to be associated with inhibiting the c-Jun N-terminal protein kinase signaling pathway.

Objective To establish analysis methods of two-dimensional(2-DE)gel electrophoresis for human osteosarcoma.Methods A series of methods,including Immobilized pH gradient were used as ID.Some applications,such as sample preparation used as choice of IPG gel,were improved.Coomassie brilliant blue staining,ImageMaster 2D Elite 3.01 analysis software,MALDI-TOF/ TOF MS and SWISS-PROT database serching were used to separate and indentify the proteins of human osteosarcoma.Results The good use pattern including repetitive experiments showed that in three experiments,the amount of protein spots of the same team sample deviates from the relative standard as following,the average of variation coefficient(%) : 23.00?10.11 and 20.33?9.90;and the range of variation coefficient(%) were:3.80-6.89 and 2.706.89 from osteosarcoma and normal group respectively.The isoelectric point and molecular weigh of the same protein spots in three experiments deviated from relative standard as following:(8.93%?1.17)%,(10.16?2.02)%,(10.87?3.86)%,respectively.Therefore,better resolution and repetitive 2-DE atlas were obtained.The proteins from 11 pairs of sample were analysed by mass spectrometry,9 identified proteins(transthyretin precursor, Triosephosphate isomerase,slow skeletal muscle,cardiac muscle Troponin T,Cofilin-1,Myosin light chain 1,Calgranulin B,Heat-shock protein,Annexin A5, Fanconi anemia group D2 proteins) were more abundant in osteosarcoma tisstues and 2 proteins manganese SOD and carbonic dehydratase appeared down-regulation in osteosarcoma tisstues.Conclusion This optimized 2-DE map is an important tool for further study on osteosarcoma,and these identified proteins were related-proteins with osteosarcoma.It is suggested that the changes of the proteome are involved in the pathology processe of osteosarcoma.