Objective To investigate the effect of autophagy on acquired secondary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKI) in patients of non-small cell lung cancers (NSCLC). Methods Ninety-eight patients with pathological confirmation of lung adenocarcinoma were selected, and they were treated with EGFR-TKI. Among them, 49 patients were sensitive to EGFR-TKI, (EGFR-TKI sensitive group), and the others were resistant (EGFR-TKI resistant group). The expressions of mTOR, Beclin-1 and LC3 were detected by immunohistochemistry, reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot method. Results The immunohistochemistry results showed that the Beclin-1 and LC3 positive expression rates of tumor tissue in EGFR-TKI resistant group were significantly lower than those in EGFR-TKI sensitive group:24.49%(12/49) vs. 83.67%(41/49) and 22.45% (11/49) vs. 85.71% (42/49), and the mTOR positive expression rate was significantly higher than that in EGFR-TKI sensitive group: 77.55% (38/49) vs. 20.41% (10/49), and there were statistical differences (P<0.05). The RT-PCR results showed that the Beclin-1 and LC3 positive expressions of tumor tissue in EGFR-TKI resistant group were significantly lower than those in EGFR-TKI sensitive group: 0.723 ± 0.029 vs. 2.542 ± 0.104 and 0.886 ± 0.034 vs. 2.234 ± 0.164, and the mTOR positive expression was significantly higher than that in EGFR-TKI sensitive group:2.142 ± 0.132 vs. 0.638 ± 0.031, and there were statistical differences (P<0.05). The Western blot results showed that the Beclin-1 and LC3 positive expressions of tumor tissue in EGFR-TKI resistant group were significantly lower than those in EGFR-TKI sensitive group:0.315 ± 0.037 vs. 1.226 ± 0.017 and 0.420 ± 0.016 vs. 1.023 ± 0.014, and the mTOR positive expression was significantly higher than that in EGFR-TKI sensitive group: 0.986 ± 0.032 vs. 0.282 ± 0.021, and there were statistical differences (P<0.05). In EGFR-TKI sensitive group and EGFR-TKI resistant group, the Beclin-1 and LC3 showed positive correlation, and the Beclin1 and LC3 showed negative correlation with mTOR. Conclusions The signaling molecules of autophagy play an important role in secondary resistance to EGFR-TKI in patients of NSCLC. As a regulation mechanism of autophagy, mTOR takes part in the procedure of resistance to EGFR-TKI, and give a new biological marker of the predication of drug resistance. Meanwhile, it gives a new target of drug resistance reversal and individualized treatment.

Objective:To evaluate the effect of semiconductor lasers irradiation after routine root canal preparation on root cannal seal.Methods:60 Single-rooted freshly extracted human teeth were randomly divided into 6 groups(n=10).The crowns were removed at the cementoenamel junction and the roots were endodontically prepared with conventional methods.The roots in groups A and B were irradiated with 1 W semiconductor laser for 20 s,in group C and D were ultrasonically washed for 1 min,in group E and F without any treatment were used as the controls.Then all the roots were filled by vertical condensation of warm gutta-percha.The root cannal seal was evaluated with microleakage measurement.The data was analyzed by ANOVA.The teeth of group B,D and F were sectioned and examined under scanning electron microscope(SEM).Results:The microleakage(mm) of group A,C and E was 1.70±0.82,2.02±0.40 and 4.56±2.72 respectively(A vs E,P<0.01;C vs E,P<0.05;A vs C,P>0.05).SEM observation showed the melting,narrowness or closure of most dentinal tubules in group B,past and/or gutta-percha in the most dentinal tubules of group D.Conclusion:Semiconductor laser irradiation prior to root cannal filling can promote the effects of cannal seal.

Background The scarring of conjunctival filtering blebs after glaucomatous surgery is a leading cause of operation failure.Exploring the balance between matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of matrix metalloproteinase-2 (TIMP-2) in conjunctival filtering bleb is very important for the study on pathogenesis of postoperative scarring.Objective This study was to evaluate the role of MMP-2 and TIMP-2 in wounding healing of subconjunctival tissue after filtering surgery in rats.Methods Sixty-three clean male SD rats were divided into normal control group and postoperative 1-day,3-day,5-day,7-day,14-day and 28-day group.The drainage tube was monocularly implanted into the anterior chamber of the rats to establish the filtering surgery models,and the operative response of the eyes was examined under the slit lamp microscope.The animals were sacrificed in corresponding time points,and the frozen sections of eyeballs were prepared,and the expressions of MMP-2 and TIMP-2 in conjunctival and subconjunctival tissues were detected using immunofluorescence technique.Western blot was employed to assay the dynamic expressions of MMP-2 and TIMP-2 proteins in conjunctival and subconjunctival tissues in different groups.The use and care of the rats complied with the Instruction Notions with Respect to Care for Laboratory by State Ministry of Science and Technology.Results Filtering blebs were formed in all the operated eyes 1 day after surgery and remained for 7-17 days,with the average survival time of 12 days.Western blot assay revealed that the expression levels of MMP-2 protein in the filtering blebs of the normal control group were 121.67 ±4.37,and the expressions were gradually elevated from the postoperative 3-day group (183.67±5.61) until the postoperative 7-day group (230.50±8.48) and then gradually declined till the postoperative 28-day group (172.33 ± 8.43),showing a significant difference among the groups (F=280.18,P＜0.05).In addition,the expression levels of TIMP-2 protein in filtering blebs of the normal control group were 102.50 ±6.25.The expression levels were gradually raised in postoperative 3-day group (162.67±7.00) and peaked in the postoperative 5-day group (232.00± 11.03),and then the levels gradually reduced till the postoperative 28-day group (150.50±6.41),with a significant difference among the groups (F =145.34,P ＜ 0.05).Immunofluorescent staining showed that MMP-2 and TIMP-2 were weakly expressed in the conjunctival epithelium of the normal rats,while in the operated rats,strong fluorescence for MMP-2 and TIMP-2 was seen in both conjunctival epithelium and subconjunctival tissues of filtering blebs.Conclusions The expression trends of MMP-2 and TIMP-2 in the filtering blebs are consistent to the fibrosis process of conjunctival tissue,indicating that MMP-2 and TIMP-2 participate in the scar formation of conjunctival filtering bleb after glaucoma filtering surgery.

OBJECTIVEThis study investigated the role and mechanism of calcineurin (CaN)-nuclear factor of activated T cells (NFAT) pathway in the myoblast apoptosis induced by cyclic tensile strain.

METHODSMyoblasts were cultured using an in vitro-mechanical stimulation model and imposed with tension for different hours with a multi-channel cell stress loading system. Cyclosporine (CsA) was used as CaN inhibitor to clarify the role of CaN in the apoptosis induced by cyclic stress. Hochest 33258 staining and flow cytometry detection were performed to detect the apoptotic cells. Real-time polymerase chain reaction was conducted to detect the mRNA expression of CaN and NFAT. Protein levels of NFAT3 were evaluated by Western blot.

RESULTSThe apoptosis rate increased with the extension of loading time. The mRNA expression of the CaN subunits, CnA and CnB, and the protein levels of NFAT3 also increased. When the myoblasts were incubated with CsA, the apoptosis rate decreased, the mRNA expression of CnA and NFAT3 significantly decreased, and the NFAT3 protein expression levels became significantly lower than those of the groups without CsA.

CONCLUSIONContinuous cyclic tensile stress can induce myoblast apoptosis. The CaN-NFAT signaling pathway may be involved in the cyclic stretch-induced apoptosis of myoblasts.

Apoptosis ; Calcineurin ; genetics ; Cyclosporine ; Flow Cytometry ; Myoblasts ; physiology ; NFATC Transcription Factors ; metabolism ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; T-Lymphocytes

Objective To evaluate the changes in cholinergic anti-inflammatory pathway in hippocampi global in aged rats with cerebral ischemia/reperfusion (I/R ) injury .Methods One hundred and twenty male Sprague-Dawley rats , aged 18-22 months ,weighing 450-600 g ,were randomly divided into 2 groups ( n= 60 each):sham operation group (group S) and global cerebral I/R group (group I/R) .The animals were anesthetized with intraperitoneal 10% chloral hydrate 0.4 ml/100 g .Global cerebral I/R was induced by 4-vessel occlusion method described by Pulsinelli .Fifteen rats were sacrificed at 1 ,3 ,5 and 7 days of reperfusion ,and brains were removed for determination of neuronal apoptosis and expression of α7 nicotinic acetylcholine receptor (α7nAChR ) , choline acetyltransferase (ChAT ) ,tumor necrosis factor-α(TNF-α) and interleukin-1β(IL-1β) in the hippocampal CA1 region .The apoptosis rate was calculated .Results Compared with group S ,the apoptosis rate was increased and the expression of α7nAChR ,ChAT ,TNF-αand IL-1βwas up-regulated in group I/R ( P<0.05 or 0.01 ) . The expression of α7nAChR and ChAT was up-regulated gradually during reperfusion and peaked at 5 day of reperfusion ( P< 0.05 ) .Conclusion Global cerebral I/R injury can activate cholinergic anti-inflammatory pathway in aged rat hippocampi ,and the activation of this pathway is the endogenous mechanism of inhibition of excessive inflammatory responses in brain tissues .

BACKGROUND:Endoplasmic reticulum stress participates in the occurrence and development of many diseases, such as atherosclerosis, diabetes, and Alzheimer’s disease. GRP78 is a marker of endoplasmic reticulum stress. The expression of GRP78 reflects the degree of endoplasmic reticulum stress.
OBJECTIVE:To investigate the effect of cyclic stretch on GRP78 expression of L6 rat myoblasts, and to identify the relationship between cyclic stretch and endoplasmic reticulum stress.
METHODS:In vitro culture-tensile stimulation models of myoblasts of L6 rats were established successful y. The expression of GRP78 of myoblasts exposed to cyclic stretch was determined by reverse transcription-PCR and western blot assay. Stretch groups were subjected to 15%surface elongation at a frequency of 10 cycles per minute, over a period of 1, 6, 12 and 24 hours. cells were simultaneously seeded on a plate in the control and experimental groups with no stimulation.
RESULTS AND CONCLUSION:The expression of GRP78 mRNA was continuously elevated over time after stretched treatment, and significant differences were detected as compared with the control group (P<0.05). GRP78 protein expression began to increase at 1 hour after stretched treatment, was significantly increased at 6 hours, peaked at 24 hours, and significant differences were visible as compared with the control group (P<0.05). In conclusion, cyclic stretch induced the occurrence of endoplasmic reticulum stress, which was enhanced with prolonged time. However, prolonged stretch caused severe endoplasmic reticulum stress and leaded to apoptosis of myoblasts.

BACKGROUND:The adaptive reconstruction of maxil ofacial muscles would happen when functional orthopedic treatment is done to cure micromaxil ary deformity. The myoblast is the main responder in the process of adaptive reconstruction, and cyclic stretch can induce apoptosis of myoblasts. Caspase-9 is an important factor in the mitochondrial apoptosis pathway.
OBJECTIVE:To investigate the expression of Caspase-9 in different cyclic stretch.
METHODS:Based on myoblasts cultured in vitro-mechanical stimulation model, the rat L6 myoblasts were loaded stretch for 1, 6, 12 and 24 hours through multi-channel cellstress loading system, while the control group received no stretch. The morphological change and growth of myoblasts were observed under inverted phase contrast microscope;the expression of the mRNA and protein of Caspase-9 were detected by RT-PCR and western blot analysis, respectively.
RESULTS AND CONCLUSION:Under inverted phase contrast microscope, the rat L6 myoblasts at cyclic stretch maintained a good growth state and biological characteristics;there was no celldegeneration;and the loss rate was extremely low, which could demonstrate that myoblast in vitro-mechanical stimulation model was established successful y. The results of RT-PCR and western blot analysis showed that, the expression of Caspase-9 mRNA and Cleaved Caspase-9 protein was significantly increased as the loading time prolonged, and the expression of Procaspase-9 protein was significantly decreased as the time. We can conclude that Caspase-9 is involved in the mechanical signal transduction of cyclic stretch.

Objective To study the effects of resveratrol on acute gouty arthritis.Methods According to random number table,thirty-six Wista rats were randomly divided into six groups:the control group,the model group,the colchicine treatment group,the high dose resveratrol group,the medium dose resveratrol group,the low dose resveratrol group.They were administrated with normal saline,colchicine suspension,high dose resveratrol,medium dose resveratrol,low dose resveratrol for 7 days once daily.Acute gouty arthritis rats models were established on the fourth day by injecting monosodium urate solution (concentration of 25 mg/ml,0.05 ml) into the ankle joint,while 0.05 ml normal saline was injected into the joint of the control rats.Seventy-two hours after the model was established,the synovial fluid were collected from ankle joints for the measuring of the level of IL-1β,CXCL10.NF-κB p65 protein levels were assayed with Western blotting.The joint synovia were fixed for histopathological examination with 10％ fromaldehyde.Variance analysis were used.Results The values of IL-1β in the low,medium and high doses of resveratrol were (9.63±0.71),(7.67±0.48) and (6.66±0.29) pg/ml respectively,being significantly lower than that in the model group (11.85±0.45) pg/ml (P＜0.05).The values of CXCL10 in the medium and high doses of resveratrol were (86.8±2.9) and (89.6±5.5) pg/ml respectively,being significantly lower than that in the model group (110.1±4.0) pg/ml (P＜0.05).The Western blotting results showed that the expression of NF-κB p65 protein in the high dose resveratrol group decreased evidently than that in the model group.The histopathological examination showed that resveratrol might reduce joint edema,decrease inflammatory cells infiltration in acute gouty arthritis.Conclusion The levels of IL-1β,CXCL10 and the expression of NF-κB p65 protein are elevated in acute gouty arthritis.The resveratrol can effectively control acute gouty arthritis attacks,and its efficacy is dose dependent.

Objective To evaluate the changes in 5'-adenosine monophosphate-activated protein kinase (AMPK) signal transduction pathway in hippocampal neurons of aged rats during transient global cerebral ischemia/reperfusion (I/R).Methods Ninety-six aged male Sprague-Dawley rats aged 18-22 months,weighing 450-600 g,were randomly allocated to one of two groups (n=48 each):sham operation group (group OS) and transient global cerebral I/R group (group OTIR).Ninety-six yong male Sprague-Dawley rats aged 3 months,weighing 200-250 g,were randomly divided into 2 groups (n=48 each):sham operation group (group AS) and transient global cerebral I/R group (group ATIR).The global cerebral I/R was produced by 3 min four-vessel occlusion followed by reperfusion according to Pulsinelli.On 3,5 and 7 days of reperfusion,12 rats in each group were chosen and sacrificed.Their brains were removed and hippocampal CA1 region was dissected for detection of neuronal apoptosis (by TUNEL) and expression of phosphorylated AMPKα (p-AMPKα) (by Western blot).The apoptotic rate (AR) was calculated.Results Compared with OS group,the AR was significantly increased and the expression of p-AMPKα was up-regulated at each time point in OTIR group,and the AR was significantly decreased and the expression of p-AMPKα was down-regulated at each time point in AS group (P ＜ 0.05).Compared with AS groupthe AR was significantly increased at each time point and the expression of p-AMPKα was up-regulated on day 3 and 5 of reperfusion in ATIR group (P ＜ 0.05).The AR was significantly lower at each time point and the expression of p-AMPKα was down-regulated on day 5 and 7 of reperfusion in ATIR group than in OTIR group (P ＜ 0.05).Conclusion Transient global cerebral I/R can activate AMPK signal transduction pathway in hippocampus of aged rats.The activation of AMPK signal transduction pathway is stronger and the cerebral I/R injury is more severe in aged rats than in young rats.

Objective To study the expressions of Cx43,CD105 and VEGF in HBV related HCC tissues and the relationships between Cx43 expression and recurrence and prognosis after cancer radical resection in HCC patients stratified by serum AFP levels. Methods The expressions of Cx43,CD105,VEGF in 234 HBV related HCC tissues were examined by tissue microarray and two-step methods of PV-6000 of immunohistochemistry and the expressions of Cx43 in 20 frozen HCC specimens were examined by RT-PCR. Results Cx43 in HCC tissues was positive as examined by both immunohistochemistry and RTPCR methods.Positive Cx43 expression is correlated with lower early recurrence ( Log Rank P =0.001 ),longer disease free survival (Log Rank P =0.026 ) and overall survival( Log Rank P =0.000 ) as showed by the Kaplan-Meier analysis in patients with AFP ＜ 400 μg/L. The expression of Cx43 is an independent prognostic factor.The positive expression of Cx43 related with lower positive expression of CD105 and VEGF (P =0.018,0.023 ),and correlated with histological differentiation (P =0.002),the number of focus (P =0.033 ),blood vessel tumor embolism ( P =0.029 ). Conclusions The expression of Cx43 is correlative with the expression of CD105 and VEGF,and is predictive of HCC early recurrence and poor prognosis after radical hepatectomy in HBV related HCC patients with serum AFP ＜ 400 μg/L.