Objective Through observation of the expression and activity of extracellular regulated protein kinase 5 (ERK5) and its relationship with the learning and memory ability in rats with chronic fluorosis,to further study the pathogenesis of chronic fluorosis in nervous system.Methods Thirty SD rats were divided into 3 groups according to body weight by means of a random number table (10 rats in each group,half male and half female).The rats in control group were fed with free drinking tap water containing less than 0.5 mg/L fluoride (NaF);the rats in low fluoride group with 10.0 mg/L fluoride;the rat in high dose fluoride group with 50.0 mg/L fluoride.After 6months of experiment,rat brain tissue was took,mRNA expression level of ERK5 was detected by real-time fluorescence quantitative PCR (real-time PCR),protein expression level and activity of ERK5 were detected by Western blotting;the learning and memory ability of rats with chronic fluorosis were detected by Morris water maze test.Results The rat in groups exposed to fluoride exhibited different degrees of dental fluorosis and the fluoride content in urine of rats increased gradually with increase of fluoride doses (F =164.10,P ＜ 0.05).The protein levels of phosphor-ERK5 in the control group,low fluoride group and high fluoride group were 0.13 ± 0.03,0.29 ± 0.10and 0.43 ±0.17,respectively,the difference was statistically significant (F=11.96,P＜ 0.05),and low fluoride group and high fluoride group were higher than control group (all P ＜ 0.05).The total protein levels of ERK5 in control group,low fluoride group and high fluoride group were 0.32 ± 0.11,0.37 ± 0.13 and 0.49 ± 0.16,respectively,the difference was statistically significant (F =3.45,P ＜ 0.05),and high fluoride group was higher than control group (P ＜ 0.05).The expression of ERK5 mRNA in rat brains between groups was not significantly different (F =0.81,P ＞ 0.05).The second,third,and forth days of directional navigation experiment,the time of escape latency and the number of crossing the platform between groups were statistically significant (H =28.20,29.90,26.47,27.23,35.34,27.62,all P ＜ 0.01);the fifth day of space exploration experiment,the difference of the time of the first crossing platform and the number of crossing the platform between groups were statistically significant (H =31.41,30.80,all P ＜ 0.01);the protein level of phosphor-ERK5 in brain tissue of rats was negatively correlated with the number of the first crossing platform (r =-0.470,P ＜ 0.01),while positively related to escape latencies at the fifth day of the test (r =0.591,P ＜ 0.01).Conclusion The changes of ERK5 signaling pathway in rat brain tissue caused by chronic fluorosis are found,which are related to the decrease of leaming and memory ability of animals with chronic fluorosis.

Objective To observe the expression of neural nicotinic acetylcholine receptor subunit α3 (α3nAChR) and extracellular regulated protein kinases (ERK1/2),c-Jun N-terminal kinase (JNK),p38 kinases of mitogen-activated protein kinase (MAPK) pathway in human neuroblastoma cell line SH-SY5Y overexposed to fluoride,and try to investigate the molecular mechanism of cell damage caused by overexposure of fluoride.Methods The SH-SY5Y cell with low expression of α3nAChR suppressed by silence interference RNA served as α3nAChR silence group;the normal SH-SY5Y cell served as control group,and the effect of silencing of αt3nAChR gene in SHSY5Y was detected by Western blotting and real-time PCR;SH-SY5Y cell was treated with different concentrations of fluoride (0.000,0.005,0.050,0.500,1.000,2.500,5.000 mmol/L),the safe concentration of fluoride in SHSY5Y cell was detected by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay;the SH-SY5Y cell of control group and α3nAChR silence group were treated with 4.000 mmol/L fluoride for 0,4,8,12,24,36,48 h according to the results of MTT assay;the expression of ERK1/2,JNK,p38 kinases of MAPK pathway in SH-SY5Y at protein levels was measured by Western blotting.Results The expression of α3nAChR mRNA (0.04 ± 0.03) and protein (12.0 ± 2.5) in α3nAChR silence group was decreased significantly compared with those of control group (1.00 ± 0.11,100.0 ± 11.3,t =24.58,28.80,all P ＜ 0.05).The viability of SH-SY5Y cell treated with 5.000 mmol/L fluoride (0.53 ± 0.15) was decreased significantly compared with that of SH-SY5Y cell treated with 0.000 mmol/L fluoride (1.05 ± 0.05,P ＜ 0.05).The increased expression of phospho-ERK1/2 was found in α3nAChR silence group and control group incubated with fluoride with time prolonged,and the expression of phospho-ERK1/2 increased significantly at time points 24,36 and 48 h (188.33 ± 7.33,200.00 ± 10.01,213.33 ± 11.55;125.33 ± 5.69,136.00 ± 4.52,155.33 ± 6.51) compared to 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-ERK1/2 was higher significantly in α3nAChR silence group than those of control group (t =9.26,7.63,5.72,all P ＜ 0.05);no change of expression of total-ERK1/2 in the two groups was found with the passage of time.The gradually increased expression of phospho-JNK was found in α3nAChR silence group and control group,among which,the expression of phospho-JNK in o3nAChR silence group at time points 12,24,36 and 48 h (154.00 ± 6.25,149.00 ± 5.57,156.00 ± 6.08,141.67 ± 2.52) and in control group at 8,12,24,36,48 h (133.33 ± 10.69,173.00 ± 4.00,175.00 ± 11.79,200.67 ± 11.93,200.33 ± 18.58) was compared to those at 0 h in the same groups (100.00 ± 0.00,100.00 ± 0.00),and the difference was significant (all P ＜ 0.05);the higher expression of phospho-JNK was found in α3nAChR silence group other than control group at 8,12,24,36,48 h (t =-4.28,-5.02,-2.89,-8.33,-6.18,all P ＜ 0.05);no change of expression of total-JNK was found in the two groups (P ＞ 0.05).The increased expression of phospho-p38 was detected in control group at time points 24,36 and 48 h (120.33 ± 4.51,122.00 ± 7.55,119.67 ± 7.57) compared to 0 h in the same groups (100.00 ± 0.00,all P ＜ 0.05),and the expression of phospho-p38 was significantly higher than that in α3nAChR silence group at the same time points (93.33 ± 9.61,94.00 ± 5.01,98.33 ± 5.69,t =-4.01,-6.73,-5.59,all P ＜ 0.05);no change of expression of total-p38 was found in the two types of SH-SY5Y cells treated with fluoride (P ＞ 0.05).Conclusion When SH-SY5Y cells are exposed to fluoride;activation of ERK1/2 may be not depend on α3nAChR;α3nAChR may have protected the cell from apoptotic injury caused by activation of JNK pathway,and the activation of p38 may be depend on nAChRα3.

BACKGROUND:There remains controversy that whether patel ar resurfacing in total knee arthroplasty and whether patel ar chondromalacia has influence on clinical outcomes.
OBJECTIVE:To evaluate anterior knee pain, clinical function and effects of patel ar chondromalacia on clinical outcomes after total knee arthroplasty without patel ar resurfacing for degenerative osteoarthritis.
METHODS:Clinical data of 162 patients (162 knees) with degenerative osteoarthritis undergoing total knee arthroplasty without patel ar resurfacing from June 2008 to February 2010 were retrospectively analyzed. Outerbridge classification was used for patel ar chondromalacia, Visual Analogue Scale for anterior knee pain, and the Knee Society clinical scoring system for clinical function.
RESULTS AND CONCLUSION:The incision of al patients reached stage-I healing. The patel ar chondromalacia:grade I in 18 patients, grade II in 36 patients, grade III in 62 patients, and grade IV in 35 patients. At the final fol ow-up, there were six (4.0%) patients with anterior knee pain, including four cases of mild pain and two cases of moderate pain, no severe pain. The mean Knee Society clinical scoring system scores and patel ar score were obviously elevated. Outerbridge classification did not affect the incidence of anterior knee pain after replacement (χ2=0.42, P=0.94), the Knee Society clinical scoring system score (knee score:F=1.83, P=0.14;functional score:F=0.56, P=0.64) and partel ar score (F=0.78, P=0.51). These data suggested that total knee arthroplasty without patel ar resurfacing for degenerative osteoarthritis can obtain satisfactory clinical outcomes, and the patel ar chondromalacia may not affect the clinical outcomes.

Objective To observe the effects of fluorosis on the levels of endogenous cystathionine beta-synthase (CBS) and hydrogen sulfide (H2S) in rats.Methods According to body weight,forty-eight Sprague-Dawley rats (body weight 105-180 g) were divided into three groups by a random number table(16 rats in each group,half male).Fluorine contents of the feed in control group,low-fluoride group and high-fluoride group were 9.80,15.40 and 23.80 mg/kg.After 6 months of fluorine exposure,the fluorine contents of urine and bone were determined by the method of fluorine ion-selective electrode ; H2S levels in serum and brain and the activity of CBS in brain were detected by methylene blue; and protein expression of CBS was detected by Western blotting.Results Compared with control group,dental fluorosis was found in rats of low-fluoride and high-fluoride groups.The differences of fluorine contents of urine and femur were statistically significant between groups(F =65.16,67.93,all P ＜ 0.05).The urinary and femoral fluorine in low-fluoride groups [(5.25 ± 0.45)mg/L,(1 196.54 ± 72.78)mg/kg] and high-fluoride groups[(13.17 ± 0.98)mg/L,(2 656.61 ± 170.12)mg/kg] were higher than those of control groups [(3.64 ± 0.20)mg/L,(870.71 ± 71.51)mg/kg,all P ＜ 0.05],and the increases were in a dose-dependent fashion(all P ＜ 0.01).The differences of H2S contents in serum and brain were statistically significant(F =4.83,1 456.13,all P ＜ 0.05).The H2S content in serum was higher in high-fluoride group [(17.64 ± 2.38) μ mol/L] than that of the control group [(10.29 ± 0.74) μ mol/L,P ＜ 0.01].The H2S contents in brain were higher in the low-fluoride [(364.74 ± 2.06)μmol/L] and high-fluoride groups [(513.43 ± 4.18) μmol/L] than those of the control group[(314.94 ± 0.72)μmol/L,all P ＜ 0.01],and the increase was in a dose-dependent fashion (P ＜ 0.01).The difference of CBS activity was statistically significant between groups (F =760.63,P ＜ 0.01).The CBS activities were lower in low-fluoride [(438.90 ± 2.83) mmol· kg-1· min-1] and high-fluoride groups [(529.83 ± 2.37)mmol· kg-1· min-1] than those of the control group [(596.33 ± 2.75) mmol · kg-1· min-1,all P ＜ 0.01],whereas the protein expression of CBS in brain in high-fluoride group (1.49 ± 0.08) was higher than that of the control group (1.19 ± 0.06,P ＜ 0.05).Conclusion Chronic fluorosis can affect the levels of endogenous CBS and H2,S,and the increases are in a dose-dependent fashion in addition to CBS activity.

OBJECTIVE:To establish a method for the 5 main components (original syringin,chlorogenic acid,eleutheroside E,isofraxidin and quercetin-3-rhamnoside) in the fruits and roots of wild Acanthopanax senticosus. METHODS:UPLC was per-formed on the column of Waters ACQUITY UPLC HSS T3 with mobile phase of acetonitrile-0.3% phosphoric acid (gradient elu-tion)at a flow rate of 0.2 ml/min. Detection wavelength was 300 nm,column temperature was 30 ℃,and injection volume was 10μl. RESULTS:The linear range was 24.56-184.2 μg/ml for syringin(r=0.9993),18.454-138.405 μg/ml for chlorogenic acid(r=0.9993),8.416-63.12 μg/ml for eleutheroside E (r=0.9997),3.286-24.645 μg/ml for isofraxidin (r=0.9993) and 2.522-18.915μg/ml for quercetin-3-rhamnoside(r=0.9998);RSDs of precision,stability and reproducibility tests were lower than 1%;recover-ies were 99.14%-100.50%(RSD=0.48%,n=6)for syringing in the fruits of A. senticosus、99.03%-100.45%(RSD=0.50%,n=6) for chlorogenic acid in the fruits of A. senticosus、99.22%-100.44%(RSD=0.44%,n=6)for eleutheroside E in the fruits of A. sen-ticosus、99.80%-100.80%(RDS=0.44%,n=6)for isofraxidin in the fruits of A. senticosus、99.76%-101.10%(RSD=0.51%,n=6) for quercetin-3-rhamnoside in the fruits of A. senticosus;99.21%-101.20%(RSD=0.73%,n=6)for syringing in the root of A. senti-cosus、99.81%-101.20%(RSD=0.52%,n=6)for chlorogenic acid in the root of A. senticosus、100.00%-101.50%(RSD=0.62%, n=6)for eleutheroside E in the root of A. senticosus、99.22%-100.40%(RSD=0.47%,n=6)for isofraxidin in the root of A. senti-cosus. CONCLUSIONS:The method is simple and stable with good reproducibility,and can be used for the simultaneous determi-nation of original syringin,chlorogenic acid,eleutheroside E,isofraxidin and quercetin-3-rhamnoside in the fruits and root of wild A. senticosus.

Objective To investigate the influence of chronic fluorosis on protein kinase Cβ (PKCβ)/p66shc signal pathway in the brain of rats,and reveal the molecular mechanism of brain damage.Methods According to body weight by the random number table method thirty SD rats were divided into three groups of 10 each (half females and half males),the normal control group [less than 0.5 mg/L of fluorine (prepared with NaF) in drinking water],low fluoride exposure group (10.0 mg/L fluorine),and high fluoride exposure group (50.0 mg/L fluoride).The experiment period was 6 months.The protein level of PKCβ,p66shc,phospho-p66shc and preserved ammonia acyl isomerase (Pin1) in rat brain was detected by Western blotting.The level of neuron nuclear antigen (NeuN),p66shc and phospho-p66sh in brain of rats was detected by immunohistochemistry.Results By Western blotting,the levels of PKCβ,Pin1 and phospho-p66shc protein in brain tissue in high fluoride exposure group [(193.00 ± 57.53)％,(228.21 ± 71.14)％,(201.54 ±:50.86)％] were higher than those of the normal control groups [(100.00 ± 21.24)％,(100.00 ± 40.55)％,(100.00 ± 13.35)％,all P ＜ 0.05].By immunohistochemistry,the numbers of NeuN staining in brain tissue of the rats in both high and low fluoride exposure groups [(49.50 ± 12.57)％,(65.66 ±14.58)％] were lower than that of the control group [(100.00 ± 18.32)％,all P ＜ 0.01].The level of phospho-p66shc protein in brain tissue in high fluoride exposure group [(242.66 ± 93.01)％] was higher than those of the low fluoride exposure and the normal control groups [(152.53 ± 60.65)％,(100.00 ± 25.63)％,all P ＜ 0.01].Conclusion Chronic fluorosis has increased the expressions of PKCβ,Pin1 and phospho-p66shc at protein level in brain of rats,which may be related to the molecular mechanism of brain damage resulted from chronic fluorosis.

Objective To investigate the changes of extracellular regulated protein kinases (Erk)1/2,phospho-Erk1/2,matrix metalloproteinases (MMP)-2 and MMP-9 in rats with experimental chronic fluorosis and the role of chondroitin sulfate in treatment of rat with experimental chronic fluorosis.Methods Using a group design and cell culture methods,the SH-SY5Y cells were divided into control group (culture medium with 0.0 mmol/L fluoride ion),fluoride group (fluoride ion:4.0 mmol/L) and chondroitin sulfate group (fluoride ion:4.0 mmol/L,chondroitin sulfate:0.4 g/L).The ultrastructural changes of the SH-SY5Y cells were observed through electron microscope after 24 h treatment.The SH-SY5Y cells were cultured for 72 h,the number of cells survived in three groups.were detected after stained by trypan blue.Fifteen clean grade SD rats with body weight of 100-120 g were divided into control group (tap water:fluorine content less than 0.5 mg/L),fluoride group (fluoride ion:10.0 mg/L) and chondroitin sulfate group (fluoride ion:10.0 mg/L,the rats were performed intraperitoneal injection with 0.66 mg/kg chondroitin sulfate for 5 days after intaking fluoride for 90 days) on the basis of random number table.Five rats were in each group,and the experiment was carried out for 95 days.The capability of learning and memory of rats were tested by Morris water maze test;the expression of phospho-Erk1/2,Erk1/2,MMP-2 and MMP-9 protein in brain tissue was detected by Western blotting;the expression of phospho-Erk1/2,MMP-2 and MMP-9 in hippocampus CA2 area of brain was detected by immunohistochemistry.Results More vesicles and swelling of mitochondria or endoplasmic reticulum were observed in SH-SY5Y cell treated with fluoride through electron microscope,but relatively less in chondroitin sulfate group.Survival rate and amount of SH-SY5Y treated with chondroitin sulfate [(92 ± 23)％ and (7.83 ± 1.38) × 106/ml] were significantly higher than that of fluoride group [(55 ± 2)％,(2.19 ± 1.26) × 106/ml,P ＜ 0.05].Animal experiment results showed that most rats in control group and chondroitin sulfate group used spatial direct search strategy,and the amount of this search strategy (2.20 ± 1.09,3.40 ± 1.34) was more than that in fluoride group (0.40 ± 0.54,P ＜ 0.05).The expression of phospho-Erk1/2 in brain tissue of rats in fluoride group (3.26 ± 0.88) was significantly higher than that in control group (1.53 ± 0.28) and chondroitin sulfate group (2.36 ± 0.87,P ＜ 0.05).Immunohistochemistry results showed that average gray value of phospho-Erk1/2 in chondroitin sulfate group (220.20 ± 3.09) was significantly higher than that of the control group and the fluoride group (100.00 ± 0.00,130.98 ± 1.27,P ＜ 0.05).The average gray value of MMP-2 in the fluoride group (294.52 ± 5.18) was significantly higher than that in control group and chondroitin sulfate group (100.00 ± 0.00,117.95 ± 1.55,P ＜ 0.05).The average gray value of MMP-9 protein of the fluoride group (993.64 ± 3.66) and the chondroitin sulfate group (1 167.30 ± 239) was significantly higher than that of control group (100.00 ± 0.00,P ＜ 0.05).Conclusions Erk1/2 pathway possibly maintains the stability of cell survival by regulating the expression of MMP-2 and MMP-9.Chondroitin sulfate can protective nerve cells and reduce the nervous damage caused by fluorosis to some certain extent.

OBJECTIVETo analyze the characteristics of lymph node metastasis and prognosis in patients with T1 breast cancer.

METHODSThe clinicopathological data of 354 patients with T1 breast cancer after standard treatment from March 2007 to September 2011 were collected to analyze the relationship between the clinical characteristics of T1 breast cancer, lymph node metastasis and prognostic features.

RESULTSIn the 354 patients with T1 breast cancer, 105 patients (29.7%) had lymph node metastasis, among them 73 cases (69.5%) had 1-3 lymph node metastasis, and 32 cases (30.5%) had more than 4 lymph node metastasis. The lymph node metastasis rate was 8.3% in T1a patients, 39.7% in T1b patients, and 30.4% in T1c cases (P = 0.005). Pairwise comparison showed that the difference of lymph node metastasis rate between T1a, T1b and T1c patients was statistically significant (P = 0.001 and P = 0.006, respectively). The difference of lymph node metastasis rates in T1b and T1c patients was statistically insignificant (P = 0.171). In the 354 patients of T1 breast cancer, 92 patients had vascular tumor thrombi and their lymph node metastasis rate was 71.7%, while the lymph node metastasis rate in 262 patients without vascular tumor thrombus was 14.9% (P < 0.001). The median follow-up was 49 months (range 27-81 months). 12 patients developed recurrence, and 3 patients died, one of them died of cerebrovascular accident. The 4-year disease-free survival for all patients was 96.6%, and the 4-year overall survival rate was 99.2%.

CONCLUSIONSThere is a correlation between vascular tumor thrombus, tumor size and lymph node metastasis rate. The lymph node metastasis rate is lower in T1a patients and relatively higher in T1b/c patients. Compared with patients without vascular tumor thrombus, the T1 breast cancer patients with vascular tumor thrombi have a higher lymph node metastasis rate. Generally speaking, there is a still good prognosis in patients with T1 breast cancer.

Adult ; Aged ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Breast Neoplasms ; drug therapy ; pathology ; surgery ; Carcinoma, Ductal, Breast ; drug therapy ; pathology ; surgery ; Carcinoma, Lobular ; drug therapy ; pathology ; surgery ; Disease-Free Survival ; Female ; Follow-Up Studies ; Humans ; Lymphatic Metastasis ; Mastectomy, Radical ; Middle Aged ; Neoplasm Recurrence, Local ; Neoplasm Staging ; Neoplastic Cells, Circulating ; Prognosis ; Survival Rate ; Young Adult

Objective To analyze the characteristics of lymph node metastasis and prognosis in patients with T1 breast cancer .Methods The clinicopathological data of 354 patients with T1 breast cancer after standard treatment from March 2007 to September 2011 were collected to analyze the relationship between the clinical characteristics of T 1 breast cancer , lymph node metastasis and prognostic features . Results In the 354 patients with T1 breast cancer, 105 patients (29.7%) had lymph node metastasis, among them 73 cases (69.5%) had 1-3 lymph node metastasis, and 32 cases (30.5%) had more than 4 lymph node metastasis.The lymph node metastasis rate was 8.3%in T1a patients, 39.7%in T1b patients, and 30.4% in T1c cases ( P =0.005).Pairwise comparison showed that the difference of lymph node metastasis rate between T1a,T1b and T1c patients was statistically significant (P=0.001 and P=0.006, respectively).The difference of lymph node metastasis rates in T 1b and T1c patients was statistically insignificant (P=0.171).In the 354 patients of T1 breast cancer, 92 patients had vascular tumor thrombi and their lymph node metastasis rate was 71.7%, while the lymph node metastasis rate in 262 patients without vascular tumor thrombus was 14.9%(P<0.001).The median follow-up was 49 months (range 27-81 months).12 patients developed recurrence , and 3 patients died, one of them died of cerebrovascular accident.The 4-year disease-free survival for all patients was 96.6%, and the 4-year overall survival rate was 99.2%.Conclusions There is a correlation between vascular tumor thrombus , tumor size and lymph node metastasis rate .The lymph node metastasis rate is lower in T 1a patients and relatively higher in T 1b/c patients.Compared with patients without vascular tumor thrombus , the T1 breast cancer patients with vascular tumor thrombi have a higher lymph node metastasis rate .Generally speaking , there is a still good prognosis in patients with T 1 breast cancer .

Objective To analyze the characteristics of lymph node metastasis and prognosis in patients with T1 breast cancer .Methods The clinicopathological data of 354 patients with T1 breast cancer after standard treatment from March 2007 to September 2011 were collected to analyze the relationship between the clinical characteristics of T 1 breast cancer , lymph node metastasis and prognostic features . Results In the 354 patients with T1 breast cancer, 105 patients (29.7%) had lymph node metastasis, among them 73 cases (69.5%) had 1-3 lymph node metastasis, and 32 cases (30.5%) had more than 4 lymph node metastasis.The lymph node metastasis rate was 8.3%in T1a patients, 39.7%in T1b patients, and 30.4% in T1c cases ( P =0.005).Pairwise comparison showed that the difference of lymph node metastasis rate between T1a,T1b and T1c patients was statistically significant (P=0.001 and P=0.006, respectively).The difference of lymph node metastasis rates in T 1b and T1c patients was statistically insignificant (P=0.171).In the 354 patients of T1 breast cancer, 92 patients had vascular tumor thrombi and their lymph node metastasis rate was 71.7%, while the lymph node metastasis rate in 262 patients without vascular tumor thrombus was 14.9%(P<0.001).The median follow-up was 49 months (range 27-81 months).12 patients developed recurrence , and 3 patients died, one of them died of cerebrovascular accident.The 4-year disease-free survival for all patients was 96.6%, and the 4-year overall survival rate was 99.2%.Conclusions There is a correlation between vascular tumor thrombus , tumor size and lymph node metastasis rate .The lymph node metastasis rate is lower in T 1a patients and relatively higher in T 1b/c patients.Compared with patients without vascular tumor thrombus , the T1 breast cancer patients with vascular tumor thrombi have a higher lymph node metastasis rate .Generally speaking , there is a still good prognosis in patients with T 1 breast cancer .