Objective To study the effect of chronic fluorosis on neurobehavioral development,the ability of learning and memory in offspring of rats,and the antagonistic effect of antioxidant Vitamin E (Vit E).Methods According to body weight,forty-five 1-month-old Sprague-Dawley(SD) rats of 30 females and 15 males were divided into three groups by random number table,including control group,fluorosis and Vit E antagonistic groups (15 rats with 10 females and 5 males in every group).Five months after establishing the animal model with chronic fluorosis and Vit E gavage treatments (fluoride ＜ 1,50,50 mg/L,respectively; Vit E 0,0,50 mg/kg,respectively),the rats were mated in 2:1 proportion of female:male in different groups,respectively.The fertility index of female and neurobehavioral development indicators in offspring were observed.Spatial learning and memory of offspring after birth for 30 d were evaluated by using Morris Water Maze test.Results The female fertility index exposed to fluorosis and Vit E were not significantly different as compared to those of control group(all P ＞ 0.05) ; in contrast to control groups[(6.4 + 1.8),(15.1 + 1.7)d],the time that completed the surface righting reflex [(8.1 + 1.4),(7.9 + 1.5)d] and the air righting reflex [(17.7 + 2.3),(17.2 + 1.8)d] were delayed in the offspring in fluorosis and Vit E antagonistic groups(all P ＜ 0.05) ; the completed avoidance precipice reflex and the auditory consternation did not changed significantly(all P ＞ 0.05); In addition,compared with control and Vit E antagonistic groups [(31.74 + 17.78),(34.97 ± 15.44)s,(4.50 ± 2.51),(3.80 ± 1.87)time],the average escape latency and exploration platform at five days were decreased in 30 d offspring of fluorosis group[(42.03 + 16.45)s,(2.20 + 1.62)time].Conclusion Neurobehavioral development as well as learning and memory ability in rat offspring are impaired by long-term exposure to fluoride and Vit E has exhibited an antagonistic effect to the toxicities of fluoride.

Objective To investigate the changes of expression of quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO1) at protein and mRNA levels in the brains of rats with chronic fluorosis,effect on NF-E2-related factor 2/antioxidant response element (Nrf2/ARE) signal pathway,and reveal the mechanism of brain damage induced by the disease.Methods SD rats were randomly divided to two groups of 30 each (half females and half males),e.g.the normal control group (drinking water containing less than 0.5 mg/L of fluorine) and fluoride exposed group (drinking water containing 50.0 mg/L sodium fluoride,NaF).All rats were examined at the 10 months after feeding NaF.Dental fluorosis of rats was observed; the fluoride contents in urine and bone were detected by fluoride-ion selective electrode; protein and mRNA levels of NQO1 and HO1 in brains were detected by Western blotting and quantitative real timePCR,respectively.Results The dental fluorosis was observed,and contents of fluoride in urine [(2.16 ± 0.39)mg/L] and bone [(211.07 ± 40.52)mg/kg] determined in the rats of the fluoride group were higher than those of controls [(1.70 ± 0.24)mg/L,(34.67 ± 11.15)mg/kg,t =2.11,3.23,all P＜ 0.05].The protein expression levels of NQO1 and HO1 in the brains of rats with fluorosis [(255.2 ± 14.3) ％ and (187.2 ± 11.1)％] were also higher than those of controls [(100.0 ± 12.2)％,(100.0 t 8.9)％,t =2.14,2.05,all P ＜ 0.05]; the mRNA levels of NQO1 and HO1 [(210.2 ± 9.8)％ and (154.5 ± 7.4) ％] in the rats of the fluoride group were increased as compared to those of controls [(100.0 ± 10.4)％,(100.0 ± 9.7)％,t =2.33,2.75,all P ＜ 0.05].Conclusion The expression of NQO1 and HO1 in brain of rats with fluorosis are significantly increased,which may be due to the activation of Nrf2/ARE signal pathway and may play a compensative role in enhancing antioxidant ability.

Objective To detect the expression of muscarinic acetylcholine receptors (mAChRs) at mRNA and protein levels in the brain of rats with chronic fluorosis and to reveal the role of the receptors in brain injury and learning and memory deficits.Methods Sixty healthy SD rats were divided into two groups (30 rats in each group,half males and half females) by random number table method according to body weight.In the control group,the rats were fed with drinking water containing no more than 0.5 mg/L fluoride; in the fluoride group,the rats were fed with high doses of sodium fluoride in drinking water (50.0 mg/L).Each group was fed with normal diet (6.2 mg/kg).After being exposed to fluoride for 10 months,behavioral performance was measured with Morris water maze,including the escape latency time and the numbers of crossing platforms.After being sacrificed,rat brains were taken and weighted.M1 and M3 subunits at mRNA and protein levels were detected by real-time PCR and Western blotting,respectively; the correlation between protein levels of the receptor subunits and the ability of learning and memory was analyzed.Results In fluoride group,the escape latency time [(21.68 ± 2.90)s] was significantly longer than that of control group [(6.14 ± 1.71)s,t =0.289,P ＜ 0.05]; and the number of crossing platforms [(11.62 ± 2.26)times] was significantly decreased as compared to that of control group [(19.00 ± 3.69)times,t =0.352,P ＜ 0.05].Furthermore,the mRNA expression [(17.07 ± 6.89)％,(12.25 ± 5.03)％] and the protein levels [(71.07 ± 6.89)％,(32.25 ± 4.66)％] of M1 and M3 receptors in rat brains were significantly lower as compared to those of controls [(100.00 ± 3.00)％,(100.00 ± 2.15)％ and (100.00 ± 9.01)％,(100.00 ± 10.33)％,t =0.210,0.157,0.095,0.296,all P ＜ 0.05].The escape latency and M1,M3 protein levels were negatively correlated (r =-0.683,-0.700,all P ＜0.05),and the number of space exploration and M1,M3 protein levels were positively correlated (r =0.867,0.837,all P ＜ 0.05).Conclusion Declined expression of mAChRs at mRNA and protein levels have been detected in the brain of rats with chronic fluorosis,which may be one of the main mechanism concerning the learning and memory deficits.

Objective To observe the changes of learning and memory ability and detect the expression of muscarinic acetylcholine receptor (mAChR,M receptor) at mRNA and protein levels in brains of offspring rats with chronic fluorosis,and to reveal the mechanism of the central nervous system damage.Methods Forty healthy SD rats were divided into two groups (20 in each group,half male and half female) by random number table according to body weight.In the control group,the rats were fed with drinking water containing no more than 0.5 mg/L fluoride;in the fluoride group,the rats were fed with high dose of sodium fluoride in drinking water (50.0 mg/L fluoride).Each group was fed with normal diet (6.2 mg/kg fluoride).After exposed to fluoride for 6 months,each group was mated,and brains of newborn offspring rats aged 1,7,14,21 and 28 days were taken,and expression of M1 and M3 receptors at mRNA and protein levels were analyzed by real-time PCR and Western blotting,respectively.Behavioral changes were measured by Morris water maze test at the 28 days after birth.The correlations between protein levels of M1 and M3 receptors and the ability of learning and memory at the 28 days after birth were analyzed.Results In fluoride group of the offspring rats at 28 days after birth,the escape latency time [(35.61 ± 9.00)s] was significantly longer than that in control group [(8.46 ± 3.09)s,P ＜ 0.05],while the numbers of crossing the platforms and the time of staying the platforms [(5.00 ± 2.90)times,(16.66 ± 2.79)s] were significantly decreased as compared to that of control group [(15.17 ± 3.66)times,(22.51 ± 2.66)s,all P ＜ 0.05].Furthermore,the mRNA expression and the protein levels of M1 and M3 receptors in rat brain at each phase in fluoride group were significantly decreased as compared to controls [M1 mRNA in control groups:(100.00 ± 11.00)％,(100.00 ± 17.57)％,(100.00 ± 9.14)％,(100.00 ± 7.52)％,(100.00 ± 15.78)％;M1 mRNA in fluoride groups:(20.47 ± 8.07)％,(14.00 ± 4.53)％,(16.57 ± 7.62)％,(25.56 ± 12.78)％,(16.27 ± 4.82)％;M3 mRNA in control groups:(100.00 ± 16.30)％,(100.00 ± 14.40)％,(100.00 ± 7.20)％,(100.00 ± 14.31)％,(100.00 ± 13.16)％;M3 mRNA in fluoride groups:(29.17 ± 8.00)％,(12.77 ± 2.22)％,(26.40 ± 7.20)％,(15.74 ± 3.55)％,(28.14 ± 7.53)％;M1 protein in control groups:(100.00 ± 2.24)％,(100.00 ± 8.30)％,(100.00 ± 4.61)％,(100.00 ± 13.78)％,(100.00 ± 11.72)％;M1 protein in fluoride groups:(20.47 ± 8.07)％,(14.00 ± 4.53)％,(16.57 ± 7.62)％,(25.56 ± 12.78)％,(16.27 ± 4.82)％;M3 protein in control groups:(100.00 ± 16.30)％,(100.00 ± 14.40)％,(100.00 ± 7.20)％,(100.00 ± 14.31)％,(100.00 ± 13.16)％;M3 protein in fluoride groups:(29.17 ± 8.00)％,(12.77 ± 2.22)％,(26.40 ± 7.20)％,(15.74 ± 3.55)％,(28.14 ± 7.53)％,P ＜ 0.05 or ＜ 0.01].The escape latency and M1,M3 receptors protein levels were negatively correlated (r =-0.827,-0.742,all P ＜ 0.05),and the number of space exploration and M1,M3 receptors protein levels were positively correlated (r =0.843,0.806,all P ＜ 0.05).Conclusion The expression of M receptor at protein and mRNA levels in offspring rat brains of different ages are significantly declined,which might be one of the mechanism of the decreased ability of learning and memory induced by fluoride toxicity.

Objective To observe the expression of NF-E2-related factor 2 (Nrf2) and Kelch-like ECH-associated protein-1 (Keap1) at mRNA and protein levels in the brain of rats with chronic fluorosis and to reveal the mechanism of brain damage induced by the factors.Methods Ninety SD rats were divided into three groups (30 rats in each group,half male half female) by the random number table method according to body weight.The control group was fed with normal tap-water,high-fluoride group with 50 mg/L fluoride (NaF) added in drinking water;and the high-fluoride plus vitamin E (Vit E) group with the same dose of NaF as the high-fluoride group,but giving 5 mg/kg Vit E by intragastric administration.The experiment period was 10 months.The fluoride contents in urine and bone were detected by fluoride-ion selective electrode.The protein and mRNA levels of Nrf2 and Keap1 in brain of rats were detected by Western blotting and quantitative real time PCR,respectively.The activity of superoxid dismutas (SOD) and the content of lipid peroxidation (MDA) were measured by biochemistry methods.Results Dental fluorosis was detected in high-fluoride group.The differences of fluoride contents in urine and bone were statistically significant between groups (F =6.87,182.87,all P ＜ 0.05).The urine fluoride [(2.16 ± 0.39),(2.07 ± 0.15)mg/L] and bone fluoride [(211.07 ± 40.52),(82.09 ± 28.60)mg/kg] in the high-fluoride and high-fluoride plus Vit E groups were higher than those of the control group [(1.70 ± 0.24)mg/L,(34.67 ± 11.15)mg/kg,all P ＜ 0.05].The differences of mRNA and protein levels of Nrf2 and Keap1 in brains of rats,SOD activity,MDA content were statistically significant between groups (F =654.33,432.87,447.45,398.88,68.34,68.34,all P ＜ 0.05).The mRNA levels of Nrf2 and Keap1 [(320.18 ± 6.83)％,(267.37 t 7.22)％] were increased compared to those of control group [(100.00 ±3.00)％,(100.00 ± 2.75)％,all P ＜ 0.05];the protein levels of Nrf2 and Keap1 [(283.28 ± 6.89)％,(196.32 ± 5.57)％]were also raised compared with those of control group [(100.00 ± 8.71)％,(100.00 ± 9.23)％,all P ＜ 0.05];the activity of SOD [(22.10 ± 2.10)μ,mol/kg] in brain of rats in fluoride group was significantly lower and the content of MDA [(8.63 ± 0.77) μmol/kg] was higher than those of control group [(35.05 ± 2.98),(1.25 ± 0.64) μmol/kg,all P ＜ 0.05].In high-fluoride plus Vit E group,the mRNA levels of Nrf2 and Keap1 [(243.23 ± 5.34)％,(180.54 ± 4.48)％] and the protein levels of Nrf2 and Keap1 [(210.88 ± 4.79)％,(150.68 ± 6.49)％] were lower than those of high-fluoride group (all P ＜ 0.05);the activity of SOD [(26.33 ± 1.84)μmol/kg] was significantly higher and the content of MDA [(4.88 ± 0.84)μmol/kg] was lower than that of high-fluoride group (all P ＜ 0.05).Correlation analysis showed that increased levels of Nrf2 and Keap1 were positively correlated with the level of MDA in rat brain (r =0.69,0.33,all P ＜ 0.05),but negatively correlated with the activity of SOD (r =-0.78,-0.80,all P ＜0.05).Conclusion The expression of Nrf2 and Keap1 in brain of rats with fluorosis is significantly increased and positively correlated with the content of fluoride in bone and the level of oxidative stress,whereas vit E can attenuate these abnormal changes induced by fluoride,which might be one of the mechanisms of brain damage of the disease.

Objective To detect the levels of oxidative stress in brain and serum of rats with chronic fluorosis and the antagonistic effects of vitamin E (VitE),and to reveal the role of oxidative stress in brain injury.Methods Thirty healthy SD rats were divided into three groups based on body weight by means of a random number table (10 rats in each group,half male and half female).In the control group,the rats were fed with drinking water containing less than 0.5 mg/L fluoride;in the fluoride group,the rats were fed with high doses of sodium fluoride in drinking water (50.0 mg/L) and the VitE antagonistic group were fed with the same content of fluoride in drinking water as the fluoride group,but adding VitE (50.0 mg/kg) by intragastric administration once a day.All rats were fed with normal diet (6.2 mg/kg).After exposure to fluoride for 10 months,all rats were put to death,dental fluorosis of the rats was examined and the fluoride content in bone was determined by fluoride-ion selective electrode;the activity of superoxide dismutase (SOD) was determined by the xanthine oxidase method and glutathione peroxidase (GSH-Px) by the colorimetric method,the level of malonaldehyde (MDA) by the glucosinolates barbituric acid fluorescence method and the levels of OH-,H2O2 and O-·2 in rat serum and/or brain were detected by the colorimetric method.Results In the rats of the fluoride group,fluoride content in bone was higher as compared to control [bone fluoride:(211.07 ± 48.52) vs.(33.40 ± 9.26) mg/kg,P ＜ 0.01].The activities of SOD and GSH-Px in rat brains of the fluoride group [(20.10 ± 1.98) kU/g,(28.70 ± 19.35) kU/L] were significantly lower than those of controls [(37.05 ± 3.13) kU/g,(59.63 ± 12.83) kU/L,all P ＜ 0.01],the activity of SOD in VitE antagonistic group [(26.27 ± 1.74) kU/g] was higher than the fluoride group (P ＜ 0.01);the activities of SOD and GSH-Px in rat serum of the fluoride group were significantly decreased [(11.55 ± 1.75) kU/L,(79.50 ± 19.18) U/L] than those of controls [(20.79 ± 2.43) kU/L,(170.00 ± 14.68) U/L,all P ＜ 0.01],the activity of SOD in VitE antagonistic group [(17.23 ± 0.68) kU/L] was higher than the fluoride group (P ＜ 0.01).The levels of MDA in rat brain and serum of the fluoride group [(8.84 ± 0.69) μmol/L,(1.46 ± 0.11) nmol/L] were significantly higher than those of controls [(1.27 ± 0.74) μmol/L,(0.83 ± 0.10) nmol/L,all P＜ 0.01],VitE antagonistic groups [(4.51 ± 1.13) μmol/L,(1.29 ± 0.02) nmol/L] were lower than the fluoride groups (all P ＜ 0.01).The levels of OH-,H2O2 and O-·2 in rat brains of the fluoride group [(24.24 ± 1.80) kU/g,(15.28 ± 2.97) mmol/L,(6.53 ± 0.96) U/g] were significantly higher than those of controls [(11.44 ± 1.63) kU/g,(5.28 ± 1.20) mmol/L、,(2.93 ± 0.42) U/g,all P ＜ 0.01],VitE antagonistic groups [(14.43 ± 0.76) kU/g,(8.09 ± 0.55) mmol/L,(4.41 ± 0.49) U/g] were lower than the fluoride groups (all P ＜ 0.01).Conclusions Elevated levels of oxidative stress are found in brain and serum of the rats with chronic fluorosis,which may be a main mechanism of brain injury.VitE may play an important antagonistic role in oxidative damage induced by fluoride toxicity.

Objective To study the characteristics of pharmacokinetics and pharmacodynamics of insulin dry powder inhalation and its relative bioavailability as compared with subcutaneous injection of regular insulin. Methods In this open,single-center,randomized,two-period,cross-over,euglycemic glucose clamp study,18 healthy volunteers(14 men and 4 women),aged(24.9?1.7)years,with body mass index(20.6?1.2)kg/m~2, received the insulin dry powder inhalatin(80 U)or regular insulin(15 U)subcutaneous administration.The blood samples of this study at 0,20,30,40,50,60,70,80,90,100,110,120,135,150,165,180,195, 210,225,240,270,300,330,360,390,420,450 and 480 rain were taken for serum insulin measurement, meanwhile,glucose infusion rates(GIR)were determined per 5 minutes over a period of 8 hours.Results The C_(max)were(57.9?17.8 vs 114.5?29.7)mU/L(tested vs reference preparation),T_(max)were(46.7?45.6 vs 107.8?33.7)min,GIR_(max)were(3.35?0.98 vs 5.17?1.75)mg?kg~(-1)?min~(-1)and T_(GIRmax)were(88.3?17.0 vs 151.9?34.6)min.The relative bioavailability was(10.26?2.25)%,and the relative bioefficacy was(14.33?7.26)%.Conclusion The study shows that insulin dry powder inhalation is absorbed via lungs and its action sets in earlier than that of the regular insulin injected subcutaneously.These pharmacokinetie and pharmacodynamic data may provide a reliabe guide for further clinical trial.

Inositol requiring enzyme 1 alpha (IRE1α), a widespread transmembrane protein in mammals, is an endoplasmic reticulum stress (ER stress) receptor. Among the three signaling pathways of the unfolded protein response (UPR), the IRE1α pathway is the most conservative. And there is a growing body of evidence that the occurrence and development of tumors is closely related to the over-expression of IRE1α. Therefore, the study of the IRE1α inhibitors is of great significance to the discovery of new anti-tumor drugs and has been attracting more and more attention. In the hope of providing ideas for the research of targeting IRE1α for cancer therapy, this paper reviewed the data of representative IRE1α inhibitors, including inhibitory activity, the mechanism of action, structural characteristics, and so on.

Abstract?AIM: To investigate the influence of propylene glycol mannite sulfate ( PGMS ) on the expression of tumor necrosis factor -α( TNF-α) and interleukin-1β( IL-1β) , in diabetic retinopathy by a rat model, to study the mechanism of PGMS against diabetic retinopathy, and provide a reliable theoretical and experimental evidence for the PGMS to be applied to clinical prevention and treatment of diabetic retinopathy.?METHODS: Male Wistar rats were randomized into 4 groups, normal control group, diabetic control group and PGMS in group, the PGMS in groups included the doses of 50mg/kg and 100mg/kg. 1% streptozotocin ( STZ) of 60 mg/kg was intraperitoneally injected in rats to establish the diabetic models. The PGMS with the doses of 50mg/kg and 100mg/kg were used to gavage in different groups of models for 12wk.Twelve weeks later, the animals were sacrificed and retinas were isolated. The aqueous humors and serums were taken, expressions of TNF-αand IL-1βprotein in retinas, aqueous humors and serums were detected by enzyme-linked immunosorbent assay ( ELISA) , respectively.The location and the expression of TNF-αand IL-1βprotein in retina tissue was detected by immunohistochemistry.?RESULTS: Twelve weeks after the use of PGMS, the level of blood glucose was not changed.ELISA showed that the expression of TNF-αand IL-1βprotein in serum and retina was significantly increased in diabetic control group than in normal control group(P<0.05), but in the groups which PGMS was given reduced, lower than those in diabetes mellitus( DM) group, especially as the concentration of PGMS increased ( P<0.05 ).But the levels of aqueous humor's TNF-αand IL-1βproteins in PGMS group were not reduced.Immunohistochemistry showed that the TNF -α protein was almost not expressed in normal control group. But the TNF-αprotein was highly expressed in diabetic control group. The expression mainly located in the ganglion cell layer, the inner plexiform layer, outer plexiform layer and pigment epithelium. The TNF-αprotein was weakly expressed at the group of 50mg/kg PGMS, the TNF-αprotein was almost not expressed at the group of 100mg/kg PGMS.When the normal control group was detected, the IL-1βprotein was weakly expressed in the outer plexiform layer.But the IL-1βprotein was also highly expressed in diabetic control group.The expression mainly located in the inner plexiform layer, outer plexiform layer and pigment epithelium. The IL -1βprotein was weakly expressed at the group of 50mg/kg and 100mg/kg PGMS.?CONCLUSION:PGMS can treat the diabetic retinopathy by downregulating the expressions of TNF-αand IL-1βin early diabetic retinopathy.PGMS maybe have a good control effect on early diabetic retinopathy.

Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma were widely used in strengthening spleen under different disease conditions, and were easily and often misused each other. Therefore, DNA barcode was used to distinguish Atractylodis Macrocephalae Rhizoma and Atractylodis Rhizoma from their adulterants to ensure the safe use. The sequence lengths of ITS2 of Atractylodes macrocephala, Atractylodis Rhizoma (A. lancea, A. japonica and A. coreana) were both 229 bp. Among the ITS2 sequences of A. macrocephala, only one G/C transversion was detected at site 98, and the average GC content was 69.42%. No variable site was detected in the ITS2 sequences of A. lancea. The maximum K2P intraspecific genetic distances of both A. japonica and A. coreana were 0.013. The maximum K2P intraspecific genetic distances of A. macrocephala, A. lancea, A. japonica and A. coreana were less than the minimum interspecific genetic distance of adulterants. The ITS2 sequences in each of these polytypic species were separated into pairs of divergent clusters in the NJ tree. DNA barcoding could be used as a fast and accurate identification method to distinguish Atractylodis Macrocephalae Rhizoma, Atractylodis Rhizoma, from their adulterants to ensure its safe use.
Atractylodes ; classification ; genetics ; DNA Barcoding, Taxonomic ; methods ; DNA, Plant ; genetics ; DNA, Ribosomal Spacer ; genetics ; Drug Contamination ; prevention & control ; Drugs, Chinese Herbal ; chemistry ; classification ; Molecular Sequence Data ; Phylogeny ; Quality Control ; Rhizome ; classification ; genetics