Objective:To determine the mechanism of sunitinib-induced autophagy in renal cell carci-noma cells.Methods:MTS assay was applied to detect the cell viability alteration under the treatment of sunitinib (2,8 μmol /L).The sunitinib-induced autophagy as well as cell apoptosis was measured and compared after knocking down autophagy-related protein Beclin1 and microtubule associated protein 1 light chain 3 fusion protein (LC3)by RNA interference.The transmission electron microscope was used to observe the formation of autophagosomes in ACHN cells.The fluorescence microscope was used to mo-nitor distribution and aggregation of endogenous LC3-Ⅱ.The expressions of protein such as LC3-Ⅱ,the autophagic regulation molecules protein kinase B /mammalian target of rapamycin (Akt/mTOR)and the symbol of apoptosis poly ADP-ribose polymerase (PARP)were capable to be detected by immunoblotting assay.Results:Sunitinib was able to significantly trigger cell viability loss in the renal carcinoma cell ACHN,which was both in a concentration-dependent and time-dependent manner (P <0.05 ).After reducing the autophagy by knocking down Beclin1 and LC3,the number of cleavage of PARP was in-creased remarkably,whereas there was nearly not any cleavage in the mock group.By the transmission electron microscope,there were more autophagic vacuoles in ACHN cells after being administrated with sunitininb compared with the control.And the nuclear-to-cytosol translocation as well as aggregation of LC3-Ⅱ was presented after sunitinib treatment by the fluorescence microscope,which was the proof of the enhanced autophagy.According to the immunoblotting,sunitinib was able to increase the accumula-tion of LC3-Ⅱ.At the same time,the result of sunitinib combined with chloroquine,a drug which blocked the fusion of autophagosomes and lysosomes,demonstrated that the increasing amount of LC3-Ⅱwas due to the enhanced autophagy flux by sunitinib treatment in ACHN cells.However,phosphorylation of Akt as well as mTOR was decreased at the same time.The rapamycin (mTOR inhibitor)or knocking down Akt subunits could change the sunitinib-induced LC3-Ⅱ accumulation,whereas overexpression of Akt subunits decreased the autophagic flux,indicating that Akt/mTOR was the target of sunitinib in auto-phagy.Conclusion:Sunitinib induced autophagy via suppressing Akt/mTOR pathway,and the auto-phagy was involved in apopotosis.

Objective To investigate the autophagy capacity in clear cell renal cell carcinoma tissue compared with adjacent normal tissue.Methods Sixty-nine pairs of samples from human clear cell renal cell carcinoma tissues and relatively healthy renal tissues adjacent to the tumor were collected during surgical resection.The expressions of proteins that were participating in the regulation and execution of autophagy were detected by immunohistochemisty.Electron-microscopy was also carried out for the morphometrical analysis.Results The protein expression of p-mTOR (P =0.004),P62 (P =0.000) and ULK1 (P =0.000) were up-regulated in the carcinoma tissue,while the expression of Beclin1 (P=0.000),LC3 (P =0.000) and ATG7 (P =0.000) were down-regulated,and the expression of ATG5 (P =0.349) had no signif-icant difference compared with adjacent normal tissue.Morphometrical analysis showed that the basal autophagic activity (measured by the presence of autophagy vacuole compartment) was remarkably down-regulated in carcinoma tissue,compared with adjacent normal tissue.The expression level of mTOR was correlated with P62,LC3 and ATG7,but results showed no correlation between mTOR and Beclin 1.Conclusion Our studies show a relatively reduced autophagy capacity in clear cell renal cell carcinoma,which is regulated by multiple autophagy-related proteins such as mTOR,Beclin 1 and LC3.

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi-um (SF)(2.64 scores). In contrast, the number of punctate GFP-LC3 remained at a very low level (1.85 scores), when cells were incubated with DHT in SFA(serum-free medium+DHT). Statistical analysis showed the significant difference between SF and SFA (P<0.01). Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. ②Blockage of autophagy by 3-MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is (3.19±1.09)% and (3.01±0.33)% , respectively. Under androgen-free con-ditions, inhibition of autophagy by 3-MA could increase apoptosis significantly(10. 90±2.91%). While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of (1.16±0.52)%, which was statistically significant(P<0.01). Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.

Objective To explore the influence on isthmic spondylolisthesis (IS) from lumbar vertebras facet joint orientation (FJO) variation. Methods The lumbar vertebras of 60 patients scanned by 16-slices spiral CT were collected, 30 cases with IS was defined as isthmic group;30 cases with normal lumbar vertebras was defined as control group, the FJO at L3-4, L4-5 and L5-S1 were measured in two groups. The difference between two groups were compared at three levels, the difference between both sides of the facet joint orientation also was compared;the measured data and the data of 60 patients from foreign literature were compared at three levels. Results The FJO in isthmic group were (47.9±6.3)° ,(37.5±7.3)° , (37.9±7.7)° at the right of L3-4, L4-5, L5-S1 levels respectively,the FJO in control group were (53.1 ± 7.3)° , (40.5±6.3)° , (38.5±7.3)° respectively, the FJO in isthmic group were (48.1±6.0)° , (37.9 ± 7.4)° , (37.6 ± 7.6)° at the left of L3-4, L4-5, L5-S1 levels respectively, the FJO in control group were (52.3 ± 7.6)° , (41.6 ± 6.0)° , (38.2 ± 7.2)° respectively. The significant difference was found at L3-4 and L4-5 levels (P < 0.05), the orientation was similar at L5-S1 level (P > 0.05). The difference between the both sides FJO of lumbar vertebras was not found at L3-4,L4-5 and L5-S1 levels (P >0.05). The same as the measured data and the corresponding data from foreign literature(P > 0.05 ). Conclusions It seems possible that the coronal FJO may be the phenotypic representation of the familial etiology of IS. It is helpful for the measurement of lumbar vertebras FJO to find IS early,it is important to reduce and release the IS.

Objective: To investigate epidemiological characteristics of hospitalized children with burn injury in the author′s affiliation, so as to provide theoretical basis for developing prevention strategies of children with burn injury. Methods: Medical records of 384 and 596 hospitalized children with burn injury, aged 0 to 12-year-old, were collected respectively from January 2001 to December 2005 and January 2011 to December 2015. Percentage of children with burn injury to total hospitalized patients with burn injury in the same period of time, age, causes of injury, gender, injury month, residence, condition of first aid measures conforming to medical standard, time of admission post injury, burn degree, and operation condition of children with burn injury were analyzed. Data were processed with Mann-Whitney U test and Chi-square test. Results: From January 2001 to December 2005 and January 2011 to December 2015, percentages of children with burn injury to total hospitalized patients with burn injury in the same period of time were respectively 23.6% (384/1 626) and 25.4% (596/2 346) , with no statistically significant difference (χ²=1.653, P>0.05). Age of all children with burn injury was 1.0 (1.0, 2.0) year old from January 2011 to December 2015, obviously lower than that from January 2001 to December 2005[1.0 (1.0, 3.0) year old, Z=-3.257, P<0.01]. Ages of children with burn caused by hot liquid and electrical burn from January 2011 to December 2015 were obviously lower than those from January 2001 to December 2005 (with Z values respectively -4.248 and -2.040, P<0.05 or P<0.01). Compared with that from January 2001 to December 2005, age of children with burn caused by flame from January 2011 to December 2015 increased, with no statistically significant difference (Z=1.852, P>0.05). There was no statistically significant difference in gender of children with burn injury between the two periods of time (χ²=1.374, P>0.05). Burn injury of children in the two periods of time mainly occurred in Spring, and season of burn injury between the two periods of time was similar (χ²=1.177, P>0.05). There was statistically significant difference in residence of children with burn injury between the two periods of time (χ²=15.513, P<0.01). The number of children with burn injury of first aid measures conforming to medical standard and admission within 6 h post injury from January 2011 to December 2015 was obviously more than that from January 2001 to December 2005 (with χ² values respectively 7.434 and 43.961, P values below 0.01). Burn degrees of children with burn injury mainly were moderate in the two periods of time, and there was no statistically significant difference in burn degree and condition of operation between the two periods of time (with χ² values respectively 5.731 and 1.583, P values above 0.05). Conclusions Burn of children is a social problem. We should make great efforts on popularization of prevention and treatment about burn of children, especially children with younger age in rural areas. We should publicize standard first aid measures of burn of children and advocate admission of burn of children within 6 h post burn injury for treatment.

Objective To describe the distribution of positive lymph nodes of muscle invasive bladder cancer, and explore the relationship between positive nodes and pathological characters. Methods Pathological data from 208 consecutive cases of muscle invasive bladder cancer were collect-ed and reviewed. The correlation of tumor grade, tumor stage and lymph nodes status was analyzed. The locations and numbers of positive nodes were recorded and compared according to the specific grade or stage. Results There were 153 cases (73.6%)of G_3 tumor and 55 cases(26.4%) of G_2 tumor and none G_1 (0%)in this cohort. The case number from pT1 to pT4 was 59(28. 4%)、58 (27.8%)、48(23.0%)and 43(20.6%), respectively. The tumor grade was positively correlated with tumor stage in this cohort (r=0. 392, P=0. 000). 153 cases had been taken lymph node dissection. There was more node positive cases in pT_3 and pT_4 than that in T_1 (P=0. 001 ,P=0. 000), as well as pT_4 compared with pT_2 (P= 0. 012). The data showed that most of the positive nodes were located within the pelvic region. There was only 1 case and 1 node positive for G_1/G_2 tumor with 24.84% of node positive cases for G_3. The positive nodes involved from pelvic to proximal artery while the stage increased. Conclusions There is less chance for low grade (G_1/G_2) bladder cancer to be node posi-tive compared with G_3 ones. It is necessary to take a extensive lymphadenectomy for the patients with stage more than T_2.

Objective To explore the effect of postweaning enriched environment and citalopram treatment during juvenility on the behaviour of male rats exposed to early adverse stress. Methods The newborn pups were randomly divided into maternal separation group (MS) and non-maternal separation group (NMS). Offspring were weaned on PND22 and housed in same-rearing groups under either standard or enriched conditions or citalopram treatment until adulthood. All of them were examined by sucrose consumption test, forced swimming test (FST) and morris water maze test (MWZ). Results (1) MS had significantly less consumption of sucrose intake (ml/g)(0.013 ±0.006, n = 10), compared with the following five groups (MS + EE (0.023 ±0.012, n = 8); MS + Drug (0.027 ±0.012, n = 9); NMS (0.022 ± 0. 007, n=11);NMS + EE (0.023 ±0.007, n = 7); NMS + Drug (0.032 ±0.011),n=7)), NMS + Drug had significantly increased the radio of sucrose consumption on NMS group. (2)The immobility in FST were longer in MS-experienced groups (MS (140. 19 ± 37.01) s, n = 8); MS + EE (129.41 ±29.50)s, n = 6) ;NMS + Drug (128.83 ±26. 11)s, n = 6)) than three non-MS groups (NMS (96.28 ±35.63)s,n = 7); NMS + EE (94.17 ±24. 87)s, n = 6) ;NMS + Drug (93.00 ±34. 21)s, n = 6)). (3) MS had shorter time and shorter percentage of distance spent in target quadrant in MWZ,citalopram treatment markedly improved spatial memory on NMS group. Conclusion Maternal separation applied in newborn rats induces a broad spectrum of behavioral changes reminiscent of depressive symptoms in humans, which might be reversed to some extent by EE and antidepressant in young adults.

Objective To explore the radiosensitivity of berberine on esophageal cancer cells under hypoxia condition.Methods MTT assay and clonogenic survival assay were used to evaluate the effect of berberine on proliferation inhibition and radiosensitivity of esophageal cancer cells,respectively.Immunofluorescence was employed to examine the expression of HIF-1.The change of cell cycle distribution and cell apoptosis was assayed by flow cytometry.The expression of HIF-1 was measured by Western blot.DNA damage was detected by γ-H2AX Foci counting.Results With a clear dose and time effect,berberine inhibited cell proliferation and enhanced cell radiosensitivity(t =3.69,P＜0.05)with a sensitizing enhancement ratio(SER)of 1.42.Berberine caused a dose-dependent decrease in HIF-1 protein expression and also significantly increased the cell apoptosis in ECA-109 population(t=4.74,P＜0.05).Compared with the radiation alone group,berberine enhanced X-ray induced DNA double chain breaks(DSB).Conclusions Berberine can increase the radiosensitivity of esophageal cell line ECA-109,which may be associated with decrease of HIF-1 expression and induction of apoptosis in ECA-109 cells.

ObjectiveTo investigate the role of iNOS in the middle and late stage of pulmonary hypertension (PH) and the effect of L-Arginine(L-Arg) on these stage.MethodsThirty healthy male SD rats were randomly divided into five groups.All rats except those in control group were injected intraperitoneally (ip) with a single dose (50 mg/kg) of MCT to induce PH.Then the L3 and L5 groups were injected ip with 500 mg/kg L-Arg daily for 3 weeks and 5weeks respectively,the M3 and M5groups received a daily ip injection of the same amount of saline as L-Arg for 3 weeks and 5 weeks respectively; the same amount of saline was injected ip daily in control group for 5 weeks.Right ventricular systolic pressure(RVSP) was measured before lungs were excised for immunohistochemistry at the end of experiments. ResultsThe expressions of iNOS and elastin in M3 and M5 were higher compared to the control group,but L-Arg partly reduced the expressions of iNOS and elastin both at 3weeks and 5 weeks post-MCT.The reduction of eNOS expression in L3 and L5 groups were lower compared to M3 and M5 groups,but the eNOS expression in L3 and L5 groups were still lower than control group.ConclusionDuring the middle and late stage of PH,the expression of eNOS was decreased.The expression of iNOS was induced,which would produce numerous NO.However,these NO had no benefit on the development of PH.L-Arg could restore the balance of eNOS and iNOS,and could inhibit the development of PH,which may provide a new clues to explore the pathogenesis and treatment of PH.

Objective: To investigate the preparation procedure of Zhenjing Xiehuo granules. Methods: Using the dry extract yielding rate and the contents of liquiritin and salvianolic acid B as the indices, an orthogonal test was adopted to choose the best ex-traction and purification technology. Using the qualified ratio of granules as the index, an orthogonal test was adopted to choose the best preparation process of the granules. Results:The optimized preparation conditions were as follows:Pulvis ferri was decocted first for 60 min. The other medicines were dipped in 8-fold amount of water for 90 min, and then added into pulvis ferri extracts and decocted for 3 times with 90 min for each time. The extracts were collected and concentrated till the relative density was 1. 3 (measured at 60℃), water was added with the dilution ratio of 1:2, ethanol was added till the percentage of ethanol was 50%, and then the mixed liquid was filtered after 24 hours. After ethanol was recycled from the filtrate, the filtrate was concentrated till the relative density was 1. 3 (measured at 60℃), and then dried at 60℃. Starch as the diluent, the ratio of extract to excipient was 1:0. 8, and the wet granules were prepared with 90% ethanol as the wetting agent, dried 3 hours at 60℃ followed by size stabilization to obtain the products. Con-clusion:The optimized preparation procedure of Zhenjing Xiehuo granules is stable and feasible.