Objective:To investigate the effect of early intervention with electroacupuncture (EA) on the gut microbiota in a mouse model of post-traumatic stress disorder(PTSD).Methods:Totally 32 C57BL/6 mice were randomly assigned to the following 4 groups ( n=8 for each group): Control group, EA group, PTSD group and PTSD+ EA group.After 7 days acclimation, mice in the PTSD group and PTSD+ EA group were subjected to modified single prolonged stress (mSPS). Mice in the EA group and PTSD+ EA group received EA (2/15 Hz, 1 mA, dilatational wave, 30 min/d) on "Baihui" for 7 days. Mice in the Control group and PTSD group received false stimulation (stimulated the same acupiont without electricity) for 7 days. Seven days after the last stimulation, elevated plus maze test and fear conditioning test were conducted to observe the effect of EA on PTSD-like behavior of mice. At the same time, feces of the mice were collected for gut microbiota detection by 16S rRNA sequencing.SPSS 19.0 was used for statistical analysis.One-way ANOVA was used for multiple group comparison and Bonferrani test was done for further pairwise comparision. Results:(1) There were statistically differences in the open arm activity time of the elevated plus maze test and the immobility time in contextual and cued fear conditioning test among the four groups ( F=6.93, 5.26, 14.51, all P<0.01). In the elevated plus maze test, mice in PTSD group ((60.17±15.52) s) showed significant less time in the open arms than mice in Control group((96.37±14.62) s) and PTSD+ EA group ((86.89±15.02) s) (both P<0.05). In the fear conditioning test, mice in PTSD group ((121.99±29.67) s, (130.82±29.11) s) showed significant increased immobility time both in contextual and cued fear conditioning tests than mice in Control group((74.50±26.65) s, (39.50±23.52) s) and PTSD+ EA group ((76.77±22.60) s, (102.17±3.39) s)(both P<0.05). (2) There were no significant differences among the four groups in the alpha diversity of gut microbiota ( F=0.79-2.45, all P>0.05). (3)Correlation analysis showed that 13 gut microbiotas were negatively correlated with the immobility time in contextual fear conditioning test, 2 gut microbiotas were positively correlated with it; 7 gut microbiotas were negatively correlated with the immobility time in cued fear conditioning test, 1 gut microbiota was positively correlated with it; 3 gut microbiotas were positively correlated with time spent in open arms of elevated plus maze test. Conclusion:Early intervention with EA can improve anxiety-fear like behaviors and gut microflora disorder in PTSD model mice.

Objective:To investigate the effect of repetitive transcranial magnetic stimulation (rTMS) on the hippocampal lipidome in a rat model of chronic unpredictable stress(CUS).Methods:Twenty-four SD rats were randomly assigned to the following 3 groups ( n=8 for each group): sham group, CUS group and CUS+ rTMS group. The sham group received only sham stimulation and rats in the CUS and CUS+ rTMS group were subjected to CUS stimulation. Then, rats received 5 Hz rTMS (5 Hz, 1.26 Tesla) or sham rTMS for 7 days. After the last stimulation, all rats underwent sucrose preference test, open filed test and forced swimming test so as to observe the effect of rTMS on depressive behavior. Then, rats were sacrificed, and the levels of lipid composition in hippocampus were determined by high performance liquid chromatography mass spectrometry and analyzed by lipid search software version 4.1 and SIMCA-P 14.1.The software of SPSS 19.0 was used for statistical analysis. Univariate analysis of variance was used for comparison among groups, and Tukey test was used for multiple comparison. Results:(1)There were significant differences in open field test, sugar preference test and forced swimming test among the three groups( F=6.853-7.466, all P<0.05). In the open field experiment, the exploring time and percentage of movement distance in central area of rats in CUS group((50.72±6.38)s, (11.41±1.55)%) was significantly less than that of sham group ((86.06±7.31)s, (18.60±1.21)%) and CUS+ rTMS group((79.87±7.87)s, (16.74±1.27)%)(all P<0.05). The results of sucrose preference test showed that the percentage of sucrose intake of rats in CUS group ((37.63±6.06)%) was significantly lower than that in sham group ((68.30±6.39)%) and CUS+ rTMS group ((62.68±5.50)%)(both P<0.05) . In forced swimming test, the immobility time of rats in CUS group ((137.60±13.36)s) was significantly longer than that of sham group ((80.57±10.36)s)) and CUS+ rTMS group ((86.14±11.49)s) (both P<0.05). (2)The levels of lipid composition in hippocampus were significantly different in the three groups( F=3.826-15.440, all P<0.05). The contents of phosphatidylethanolamine (PE) ((20 850±956.56)×10 7, (24 133.33±1 242.04)×10 7), phosphatidylinositol (PI) ((788.78±136.11)×10 7, (953.65±131.26)×10 7), lysophosphatidylcholine (LPC) ((340.29±35.66)×10 7, (275.32±35.78)×10 7), creatine phosphate (CerP) ((239.65±18.14)×10 7, (293.82±38.28)×10 7), sphingosine (So) ((22.96±4.04)×10 7, (15.36±3.87)×10 7), diglyceride (DG) ((3.35±0.85)×10 7, (4.57±1.02)×10 7) and monoglyceride (MG) ((6.71±0.82)×10 7, (7.94±0.91)×10 7)in hippocampus of rats in CUS group were significantly higher than those of sham group(all P<0.05), while the phosphatidic acid(PA) ((424.52±33.38)×10 7, (509.22±42.09)×10 7) and acyl carnitine(AcCa) ((2.68±0.33)×10 7, (3.39±0.33)×10 7) decreased(both P<0.05). Compared with CUS group, the contents of PE(21 816.67±928.26)×10 7, PI(83.16±91.52)×10 7, LPC(323.59±33.91)×10 7, CerP(236.39±32.02)×10 7, So(23.35±4.46)×10 7, DG(3.16±0.85)×10 7 and MG(7.03±0.26)×10 7 in the hippocampus of CuS+ rTMS group decreased, while the contents of PA(421.55±44.28)×10 7 and ACCA(2.56±0.32)×10 7 in the hippocampus of CUS+ rTMS group increased (all P<0.05). Conclusion:The levels of glycerophospholipids, glyceroglycerides, sphingolipids, fatty acids and other lipids in the hippocampus of CUS model rats are abnormal. And the 5 Hz rTMS intervention can ameliorate the depression like behavior and the disturbances of lipid in hippocampus of CUS model rats.

Objective: To explore the association between the single nucleotide polymorphism (SNP) of rs175048 in ataxia telangiectasia mutated (ATM) gene and lung cancer susceptibility in Han population. Methods: A total of 225 cases of blood samples from lung cancer patients treated in Hospital of Traditional Chinese Medicine of Hengyang City and the Affiliated First Hospital of Nanhua University from October 2015 to August 2016 were collected as case group, and 128 cases of blood samples from healthy people were collected as the control. The polymorphisms of ATM rs175048 of above mentioned participants were detected by using the SNP sensitive On/Off Switch technique. The genotypes and allele frequencies were analyzed to compare the distribution difference between case group and control group as well as its association to the clinical features of lung cancer. Results: The genotype frequencies of AA, AT and TT of ATM rs175048 were 24.9%, 52.9%, 22.2% in case group and 42.2%, 42.2%, 15.6% in control group, respectively (all P< 0.01). Moreover, the frequencies of alleles A and T were 51.0%, 49.0% in case group, and 63.0%, 37.0% in control group (all P<0.01). Genotype TT might increase while genotype AT might decrease the risk of lung cancer. rs175048 SNP was significantly correlated with smoking, age, sex and family history (all P<0.05). Conclusion: rs175048 SNPis significantly associated with lung cancer, and TT genotype may increase the risk of lung cancer.

Objective To construct mutant recombinant vector of epidermal growth factor receptor ( EGFR) gene G719S and T790M sites associated with cervical cancer, lay the foundation for the detection of EGFR gene mutation in cervical cancer. And using it to es-tablish a molecular switch platform to detect cervical cancer EGFR gene mutations. Methods Using the wild-type recombinant plasmid as template, the mutant fusion target fragment were amplified by overlap PCR, then connect this target fragment into the vector pMD19-T. The constructed mutant recombinant plasmid was finally transformed into competent cells E.coli DH5αfurther identified by PCR with bacterial solution and genome sequencing. Establishing the molecular switch for the detection of clinical cervical cancer samples. Re-sults The G719S and T790M mutations were successfully certified by genome sequencing, and the site-directed mutant vector was successfully constructed. In addition, a molecular switch detection platform was also successfully established for the detection of cervical cancer tissue DNA. Conclusion We successfully constructed an EGFR gene mutant recombinant vector by overlap PCR technique, which providing a new technical means for gene site-directed mutagenesis. And the molecular switch detection platform was successfully established based on it, which furnishing a new method for clinical detection of EGFR gene mutations.

OBJECTIVE:To discuss the effect of early usage with Wulingjun powder on the behavior of rats with post-traumat-ic stress disorder(PTSD)and the level of interleukin-1β(IL-1β)and IL-6 in their hippocampi. METHODS:50 SD rats were ran-domized into control group(normal saline),model group(normal saline),positive control group(paroxetine,10 mg/kg)and the groups of low and high-dose(0.4,0.8 g/kg)Wulingjun powder. The rats in all groups except for control group,were exposed to single prolonged stress for the establishment of PTSD models. Corresponding drugs were given ig immediately after such establish-ment,qd,for 7 consecutive days. An open field test(the times of the rats’entering the central area and the percentage of the time the rats spent in exploring the central area within 10 min were recorded)was conducted 7 days after giving drugs;and an elevated plus-maze test(the times of the rats’in opening arms and the percentage of the time the rats spent in opening arms within 5 min were recorded)was carried out 8 days thereafter,to observe the behavior of the rats. Then the rats were sacrificed to determine the level of IL-1β and IL-6 in their hippocampi by ELISA. RESULTS:Compared to the normal group,the behavioral index of the rats in model group was significantly lower,and the level of IL-1β and IL-6 in their hippocampi was significantly higher(P<0.05 or P<0.01). Compared to model group,the behavioral index of the rats in positive control group and the group of high-dose Wuling-jun powder was significantly higher,and the level of IL-1β and IL-6 in their hippocampi was significantly lower (P<0.05),but no statistically significant difference was shown in all indexes in the group of low-dose Wulingjun powder (P>0.05). CONCLU-SIONS:High-dose Wulingjun power can improve the anxiety-like behavior of the rats with PTSD,which may be related to the re-duction in the level of IL-1βand IL-6 in the hippocampus.

Objective To investigate the effect of quetiapine (QUE) on the memory and doublecortin (DCX) expression in the hippocampus of C57BL/6 mice with cuprizone (CPZ)-induced schizophrenia in C57BL/ 6 mice.Methods 1％ dimethyl sulfoxide (DMSO) was used as a vehicle to dissolve QUE.Three group of mice,16 in each of three groups,were treated with vehicle (control group),0.2％ CPZ alone (CPZ group) or 0.2％ CPZ combined with 10 mg· kg-1 · d-1 QUE (QUE+CPZ group) for six weeks,respectively.Spatial working memory was evaluated by Y-type maze test 24 hours after the completion of the treatment period.The number of DCX positivenew neurons was calculated by immunofluorescence staining assay.The expression of Notch1 and Hes1 mRNA were detected by reverse transcription-polymerase chain reaction (RT-PCR) assay.Results (1) Y-maze test:CPZ group achieved a much lower percentage of correct alternation than control group ((22.70±6.70) ％ vs (57.69 ±6.70)％) in Y-maze test (P＜0.05).The percentage of correct alternation in CPZ + QUE group ((54.69± 10.06) ％) was significantly increased compared with CPZ group (P＜0.01).CPZ mice exhibited significant spatial working memory impairment.(2) Immunofluorescence staining:the number of DCX-positive cells in the hippocampus of the CPZ group (6342.85± 1801.72) was significantly decreased compared with that in control group (19428.57±2507.13) (P＜0.01),and it was reversed by QUE intervention (15928.57±2049.97).(3) RT-PCR:the Notch1 and Hes1 mRNA expression in CPZ group were significant lower than that in sham and CPZ + QUE group,(Notch1 (0.97±0.29) vs (0.23±0.20),P＜0.01);Hes1 (1.00±0.41) vs (0.38±0.30),P＜0.01),and there was no significant difference between sham group and CPZ + QUE group.Conclusion QUE is helpful to relieve CPZ-induced cognitive impairment and decreases expression of DCX in hippocampal,which may be related with activation of Notch1 pathway.

Objective To investigate the effects of gastrodin on neural function and the expression of myelin basic protein (MBP) and neurofilament high molecular weight (NFH) in the striatum during cerebral ischemiareperfusion in mice.Methods 36 Kunming mice were randomly divided into sham group,MCAO group and gastrodin (GAS) group.The middle cerebral artery occlusion(MCAO) was established by artery embolization.The mice in sham group were received fake surgery and saline,and the mice in MCAO and GAS group were exposed to MCAO,and received saline and GAS (100 mg/(kg · d)) injection,respectively,immediately after the operation for 7 days.On the 8th day of operation,the neurological severity scores of the mice were observed and the volume ratio of the cerebral infarction was estimated by triphenyl tetrazolium chloride (TTC) staining.Immunohistochemistry was used to detect the MBP and NF-H in the striatum.Results (1) The mice in MCAO group showed significant neurologic deficient in comparison with sham group,and the neurological severity scores of gastrodin group(3.13±0.64) were significantly higher than that(1.38±0.52) of MCAO group (P＜0.05).(2) Results of TTC staining showed that the infarction volume was obviously larger in the injured cerebral tissue in MCAO group in comparison with sham group,and the volume ratio of the cerebral infarction significantly decreased after the intervention with GAS (P＜0.05).(3) The integral optical density of MBP(272968.14±1215.23) and NF-H(12 142.73±47.16) in MCAO group decreased as compared to that((43 855.23±2434.16),(275 321.00±926.15)) in sham group and GAS group((321 531.2±2376.14),(106 135.73±598.15)) (P＜0.05).Conclusion GAS can improve neural function of mice after middle cerebral artery occlusion,and it may play an important role in protecting myelin and nerve fibers of striatum.

Objective To investigate the correlationship between depressed behaviors and interleukin-1β (IL-1β) in brain tissue in mice which are insensitive to fluoxetine,and to mimic the treatment resistant depression (TRD) in clinical condition.Methods 50 BALB/c mice were randomly divided into Control group (Control),Chronic unpredictable mild stress (CUMS) group and CUMS+fluoxetine group.Mice in Control group were raised ad libitum for 9 weeks,those in CUMS group received CUMS for 9 weeks and those in CUMS+fluoxetine group received 8 weeks' CUMS followed 1 week' s treatment with Fluoxetine(10 mg · kg-1 · d-1).At the end of the 9th week,mice in(CUMS + treatment)group were selected into antidepressant treatment-resistant mice(ATRM) as no remission and Depression Group (DM) as symptoms improved.Body mass test (BMT),open field test (OFT) and forces swim test (FST) were completed respectively in these 4 groups at the endpoint of the experiment,and the brain tissue were extracted after the tests for IL-1β Elisa test.Results (1) BMT:there was no effect of weightgain in ATRM after 1 week' s therapy with Fluoxetine.There was no difference in body-weight between ATRM ((18.56±7.56) g) and CUMS ((19.03± 8.58) g) mice,while compared with Control ((24.56±5.45) g) and DM mice ((20.12±9.17) g) ATRM and CUMS mile's body weight were significantly lower (P＜0.05).(2)OFT and FST:in OFT,there was no significant difference in of horizontal moving distance(F=0.355) either in the frequencies of entering the central zone (F=0.327) among the 4 groups;in OFT,the immobility time of ATRM ((241.50 ± ± 36.55) s) was significantly longer than that in DM ((156.00± 25.47) s) (F=13.573,P＜0.05).(3) Elisa test of IL-1β:the brain' s IL-1β serum level in ATRM ((164.90±46.70) pg/mg) was higher than those in Control ((69.68±6.56) pg/mg)),and DM ((93.09±4.65) pg/mg) (P＜0.01),while no difference with that in CUMS mice.Additionally,the depressive behaviors in ATRM showed its positive correlation with the IL-1β level in CNS (r=0.669,P=0.006).Conclusion CUMS can elicit the refractory depressive symptoms in BALB/c mice to simulate TRD' s characteristic,and the elevated level of IL-1 β within brain tissue may play an important role in the development of TRD.

Objective To investigate the effect of rosmarinic acid on the behavioral changes in enhanced single prolonged stress (ESPS) model rats and the levels of interlukin-1β (IL-1β) and interlukin-6 (IL-6) in the hippocampus.Methods 48 adult male Sprague Dawley rats were randomly divided into six groups (n =8):Control group,Control + RA (L) group,Control + RA (H) group,ESPS group,ESPS + RA (L) group and ESPS + RA (H) group.Behavioral changes of these rats were analyzed by open field test and elevated plus-maze.The levels of IL-1 β and IL-6 were measured by enzyme linked immunosorbent assay (ELISA).Results (1) Open field test showed that the number of central region entering and the fraction of time exploring in center of ESPS group were significantly reduced than that of Control group ((18.13 ± 10.15) times,(26.68 ± 10.06) ％) and ESPS + RA (H) group ((16.88 ± 8.81) times,(25.08 ± 8.52) ％) (P ＜ 0.05).And it showed no significant difference among Control + RA(L) group,Control + RA(H) group and Control group.Meanwhile,there was also no statistic difference between ESPS group and ESPS + RA(L) group.(2) Elevated plus-maze test showed that percentages of open arm entries and fraction of time exploring in open arm in reference to total number of entries into all arms and total time spent on all arms in ESPS group were significantly reduced than that of Control group((37.38 ± 8.24)％,(17.63 ±4.74)％) and ESPS + RA(H) group((33.72 ±9.49)％,(16.99 ±4.28)％) (P ＜ 0.05),but there was no significant difference between that of ESPS group and ESPS + RA(L) group(P＞0.05).It also showed no significant difference among Control + RA (L) group,Control + RA (H) group and Control group.(3) Compared with ESPS group,RA(10mg/kg) reduced the levels of IL-1β and IL-6 in the hippocampus,but RA(5mg/kg) did not have this effect.(4) Correlation analysis results showed the level of IL-1β in the hippocampus was negatively related with the ameliorated PTSD-like behaviors of ESPS exposure rats.Conclusion RA can ameliorate PTSDlike behaviors of ESPS exposure rats,and this effect may be carried out by down-regulating the levels of IL-1β and IL-6 in the hippocampus,especially the IL-1β.

Objective To investigate the effect of repetitive transcranial magnetic stimulation (rTMS) on the depressive like behaviors and expression of brain derived neurotrophic factor (BDNF),IL-1β and NF-κB of hippocampal in chronic unpredictable mild stress (CUMS) rats.Methods Thirty-two adult male rats were randomly divided into four groups (n =8):Control group,Control + rTMS group,CUMS group and CUMS + rTMS group.The sucrose preference test,forced swim test and open field test were used to evaluate depressive like behaviors for each groups.In addition,the expression of BDNF,NF-κB and IL-1 β in hippocampal were detected by western blot and ELISA after behavioral test,respectively.Results 1.The effects of rTMS on depressive like behaviors of CUMS rats:in the sucrose preference test,the sucrose preference rate of CUMS rats (0.67 ± 0.06) was significantly lower than Control group (0.91 ± 0.04),which was higher in the CUMS + rTMS group (0.83 ±0.08).In the forced swim test,the immobility time of CUMS group ((26.88 ± 11.33) s) was longer than Control group ((15.22 ± 6.75) s) and CUMS + rTMS group ((18.41 ± 6.95) s).In the open field test,both the total distance travelled and number of central area entry times of CUMS group((849.165 ± 769.01) cm,(7.42 ± 5.68))were significantly shorter ((6224.81 ± 1403.2) cm) and smaller (22.86 ± 3.72) than Control group,and those of the CUMS + rTMS were longer ((4105.57 ± 1516.92)cm) and larger (21.25 ± 3.45).All the behavioral results were statistically significant (P＜ 0.05).And of all the aforementioned behavioral parameters,there were no significant differences between Control group and Control + rTMS group(P＞0.05).2.The effects of rTMS on the hippocampal expressions of BDNF,NF-κB and IL-1β in CUMS rats:compared with Control group,the hippocampal expression of BDNF in CUMS rats was significantly decreased,while the expressions of NF-κB and IL-1β in the hippocampus were significantly increased (P＜ 0.05).Compared with CUMS group,the hippocampa expression of BD-NF in the CUMS + rTMS group was increased,and the expressions of NF-κB and IL-1β in the hippocampus was significantly decreased (P ＜ 0.05).The expressions of BDNF,NF-κB and IL-1β had no differences between Control group and Control + rTMS group.Conclusion rTMS increased the expression of BDNF,reduced the production of NF-κB and IL-1β,and alleviated depressive like behaviors in CUMS rats.