Objective To explore the influences of protein kinase Cβ (PKC3) inhibitor LY333531 on oxidative injury and apoptosis of SH-SY5Y cells induced by fluorosis.Methods The SH-SY5Y cell model of fluorosis was established,and the experiment was divided into three groups:control group [0.0 mmol/L sodium fluoride (NaF) and 0.0 μmol/L LY333531],the fluoride group (0.5 mmol/L NaF and 0.0 μmol/L LY333531),and the PKCβ inhibitor group (0.5 mmol/L NaF and 0.2 μmol/L LY333531),n =3.Flow cytometry was used to detect the changes of reactive oxygen species (ROS) and apoptosis rate,fluorescent probe technique was used to detect mitochondrial membrane potential after each group for 48 h.Results Compared with the control group [(3.32 ± 0.29) × 103,0.60 ± 0.09,(7.58 ± 1.20)％],the level of ROS [(5.99 ± 0.32) × 103] was increased,mitochondrial membrane potential (0.28 ± 0.06) was decreased,and the apoptosis rate [(18.00 ± 2.32)％] was increased in the fluoride group (all P ＜ 0.05);compared with the fluoride group,the level of ROS [(5.12 ± 0.25) × 103] was decreased,mitochondrial membrane potential (0.42 ± 0.03) was increased,and the apoptosis rate [(11.79 ± 0.70)％] was decreased in the PKCβ inhibitor group (all P ＜ 0.05).Conclusions Excess fluoride could cause oxidative damage and apoptosis in cells.PKC3 inhibitor LY333531 has a protective effect in oxidative damage and apoptosis by fluorosis.

Objective To investigate the possible pathological role of mitochondrial apoptosis pathways and its factors in fluorosis-induced apoptosis of human hepatocellular carcinoma cell strain (HepG2).Methods Under the stimulation of 1,3,6 and 9 mmol/L concentrations of NaF in vitro for 24 h (n =5),while normal control group was cultured under normal condition,the cytotoxicity was measured with MTT.The mitochondrial apoptosis inducing factor (AIF) was measured at both mRNA (n =5) and protein levels (n =6),respectively,by real-time PCR and Western blotting.The mitochondrial apoptosis related factors,such as B-cells lymphoma-2 (Bcl-2),Bcl-associated X protein (Bax),cytochrome C,caspase-9 and caspase-3 were measured at protein levels (n =6).Results After treated with 0,1,3,6 and 9 mmol/L NaF for 24 h,the cell absorbance of HepG2 cells was 0.307 ± 0.031,0.333 ± 0.028,0.230 ± 0.011,0.178 ± 0.001 and 0.152 ± 0.003,respectively,and the differences were statistically significant among groups (F =82.224,P ＜ 0.01).After treated with 3 mol/L NaF for 24 h,the mRNA level of AIF was [(153.14 ± 5.41)％] which was increased compared to the control group [(100.00 ± 4.70)％,t =-4.73,P ＜0.05].Under the same condition,the protein levels of AIF,Bcl-2,cytochrome C in cytoplasm,caspase-9 and caspase-3 were (152.16 ± 47.30)％,(171.90 ± 51.52)％,(458.00 ± 19.48)％,(527.17 ± 200.67)％ and (432.70 ±64.27)％,which were increased compared to those of the control groups [(100.00 ± 48.86)％,(100.00 ± 34.44)％,(100.00 ± 116.59)％,(100.00 ± 19.58)％ and (100.00 ± 137.16)％,t =-3.80,-3.96,-15.76,-4.64,-5.06,all P ＜ 0.05],while the protein levels of Bax and cytochrome C in mitochondrion were (24.66 ± 26.04)％,(72.99 ±45.34)％,which were decreased compared to those of the control groups [(100.00 ± 44.01)％,(100.00 ± 34.14)％,t =6.35,0.68,all P ＜ 0.05].Conclusion The mitochondrial apoptosis pathway and related factors may be involved in NaF-induced cell death in HepG2 cells.

Objective To investigate the incidence of dental fluorosis,urinary fluoride level and intelligence of children who lived in the coal-burning-borne endemic fluorosis areas and to reveal the effects of comprehensive control measures on intelligence of children in this area.Methods Children aged 8-12 who lived in coal-burning-borne endemic fluorosis areas in Bijie City of Guizhou Province were selected and divided into two groups according to the duration of comprehensive treatments given:long treatment group (Xiaba Village and Zhongtun Village,furnace stovewas changed and comprehensive control measure of health education was carried out for more than 3 years) and short treatment group(Chadi Village and Maoliping Village,stoves were improved and health education time ＜ 1 year).The children who lived in a non-fluorosis area were selected as controls in 2012.Dental fluorosis was diagnosed by the method of Dean; urinary fluoride was analyzed by the method of fluoride-ion selective electrode; and the intelligence quotient (IQ) was measured by Raven's Standard Progressive Matrices Test.Results The number of children surveyed in control group was 104,long treatment group was 298,short treatment group was 339,and the incidence rates of dental fluorosis were 0 (0/104),725％(216/298) and 85.2％ (289/339),respectively,and the incidence rates of dental fluorosis in children lived in the endemic fluorosis areas were significantly increased compared with that of control group; the difference of incidence rates between long treatment group and short treatment group was statistically significantly(x2 =15.736,P ＜ 0.01).Urinary fluoride content were (2.33 ± 0.18) and (3.03 ± 0.16)mg/L,respectively,compared with the control group[(1.34 ± 0.64) mg/L],the values in endemic fluorosis areas were significantly higher(F =306.53,P ＜ 0.01).Above average IQ of children in the control group was 97.1％ (101/104),which was significantly higher than that of long and short treatment groups; after a lengthy treatment,mental retardation detection rate was significantly lower in the low-age group,8-10 year-old ehildren(x2 =7.542,P ＜ 0.01).Urinary fluoride content was negatively correlated with the level of IQ (r =-0.553,P ＜ 0.01).Conclusions The intelligence development of children in coal-burning-borne endemic fluorosis area is significantly delayed.After a certain period of comprehensive treatment,the decreased level of cognition is inhibited and the mental retardation in the low-age group is improved.

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 understand the ABO blood type distribution of Buyi and Shui ethnic populations in Libo county of Guizhou province .Methods Totally 726 Buyi and 163 Shui individuals who were unrelated within three generations were detected ABO blood stype with glass-clotted method .Results The ABO blood type distributions in the Buyi and Shui ethnic populations both were in line with the Hardy-Weinberg equilibrium .The Buyi ethnic population ABO phenotypic distribution and gene frequen-cies were O>B>A>AB and r>q>p (r=0 .676 9 ,q=0 .176 7 ,P=0 .146 4) and the national index was 0 .842;while the Shui eth-nic population ABO phenotypic distribution and gene frequencies were O>A>B>AB and r>p>q(r=0 .522 6 ,q=0 .104 0 ,P=0 .164 7) and the national index was 1 .529 .Conclusion The Buyi and Shui ethnic populations′ABO blood type distribution in Libo county of Guizhou province is basically consistent with that of the same ethnic populations in other regions of Guizhou Province of the same ethnic groups .The genetic distance analysis prompts that the regional and ethnic differences exist in ABO blood type dis-tribution .

Objective To investigate the correlation between fibroblast growth factor receptor 2 (FGFR2) gene polymorphism and endemic fluorosis.Methods In Bijie City,Guizhou Province coal-burning-borne high fluoride areas,148 patients with fluorosis were selected as endemic fluorosis group;in non high fluoride areas of Changshun County of Guizhou Province,134 healthy people were selected as control group.Short tandem repeats (STRs)-PCR was utilized to detected the FGFR2 rs35668561 and D10S14839 microsatellite polymorphisms in endemic fluorosis cases and controls.Results FGFR2 rs35668561 461 bp (22AG)allele frequency of endemic fluorosis group (1.01％) was significantly lower than that of the control group (3.36％,x2 =5.29,P ＜ 0.05).FGFR2 D10S14839 286 bp (9GT),300 bp (16GT),310 bp (21GT) and 314 bp (23GT) allele frequency in the endemic fluorosis group were 14.53％,11.82％,16.89％ and 8.11％,in the control group were 22.01％,6.34％,8.96％ and 16.42％,the difference was statistically significant.Then 300 bp (16GT)and 310 bp (21GT)allele frequency of endemic fluorosis group was significantly higher than that of the control group (x2 =6.82,7.77,all P ＜ 0.05),and 286 bp (9GT),314 bp (23GT) allele frequency of endemic fluorosis group was significantly lower than that of the control group (x2 =5.32,9.16,all P ＜ 0.05).Conclusions FGFR2 rs35668561 and D10S14839 polymorphism are associated with endemic fluorosis.FGFR2 rs35668561 461 bp (22AG) allele may be a protective factor of endemic fluorosis.D10S14839 300 bp (16GT) and 310 bp (21GT) allele may be risk factors of endemic fluorosis,286 bp (9GT) and 314 bp (23GT) allele may be protective factors of endemic fluorosis.

Objective: To investigate the effect of lovastatin on oxidative stress and apoptosis in neurons induced by β-amyloid peptide (Aβ). Methods: Primary culture of rat hippocampal neuron was treated with Aβ oligomers alone or combined with lovastatin. The levels of OH^-, H₂O₂, O₂·^-, malondialdehyde, superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) activities were measured by biochemical methods and protein expression of caspase-3 and bcl-2 was detected by Western blot. Results: As compared with the control group, treatment of 0.5 μmol/L Aβ oligomers for 48 h led to significant increase of OH^-, H₂O₂, O₂·^- and malondialdehyde content, inhibition of SOD and GSH-PX activities, enhanced caspase-3 expression and decreased bcl-2 expression. Interestingly, these neurotoxic modifications on the levels of OH^-, H₂O₂, O₂·^- and malondialdehyde content, SOD and GSH-PX activities, and the protein expression of cleaved caspase-3 and bcl-2 were significantly attenuated when the cells were pretreated with 0.1 μmol/L lovastatin for 24 h before exposure of Aβ oligomers. Conclusion Lovastatin may play an important role in antagonizing the neurotoxicity of Aβ through a mechanism likely related to the inhibition of oxidative stress.

Objective To analyze the prognosis of children with severe mycoplasma pneumonia (MPP) and its correlation with serum SAA, PCT and SF levels, so as to provide a basis for evaluating the prognosis of children with MPP. Methods A total of 273 children with MPP admitted to our hospital from January 2020 to December 2020 were divided into mild MPP children (n=187) and severe MPP children (n=86) according to the severity of their disease. According to the prognosis, children with severe MPP were divided into survival group (n=65) and death group (n=21). Serum SAA, PCT and SF levels were determined. Pearson correlation analysis was used to analyze the correlation between serum SAA, PCT and SF levels and APACHE ⅱ score. ROC curve was used to analyze the predictive value of serum SAA, PCT and SF levels for poor prognosis of children with severe MPP. Results The levels of serum SAA, PCT and SF and APACHE II score in children with severe MPP were significantly higher than those in children with mild MPP (P<0.05). Serum SAA, PCT and SF levels and APACHE II score in death group were significantly higher than those in survival group (P<0.05). Pearson correlation analysis showed that APACHE II score was positively correlated with serum SAA, PCT and SF levels (r =0.474,0.519,0.446,P<0.05). The AUC, sensitivity and specificity of combined ROC curve analysis to predict the prognosis of severe MPP were 0.871, 85.9% and 93.6% respectively, which were higher than those of SAA, PCT and SF alone. Conclusion SAA, PCT and SF are closely related to the prognosis of severe MPP, and can be used as potential markers to predict poor prognosis of severe MPP children.

Objective To study the expression of silent information regulator (SIRT) in brains of rats with chronic fluorosis and reveal the correlation between SIRT1 and the ability of learning and memory of rats.Methods Sixty SD rats were selected and their body weight was (100 ± 20) g,according to the body mass of the rats,random number table method was used to divide rats into control group,low and high fluoride groups,experimental period was 3 and 6 months (ten rats in each experimental period,half males and half females).In control group,the rats were fed with drinking water containing no more than 0.5 mg/L fluoride;the rats in low and high fluoride groups were fed drinking water containing 5.0 and 50.0 mg/L fluoride,respectively.All of rats were fed the same standard food containing no more than 0.6 mg/kg fluoride.Three degree method was used to check the formation of dental fluorosis.Rat urinary fluoride was determined via the fluoride electrode method;Morris water maze method was used to detect the ability of learning and memory of rats (the escape latency time,the number of crossing the platform and stay time in platform quadrant);the protein and mRNA expression levels of SIRT1 were detected by Western blotting and Real-time PCR,respectively.Results In the experimental period of 3 and 6 months,no dental fluorosis was observed of rats in control group,but there were different degrees of dental fluorosis in low and high fluoride groups,especially in high fluoride group.The urinary fluorine contents [(1.60 ± 0.09),(1.91 ± 0.16) mg/L;(1.94 ± 0.19),(2.31 ± 0.18) mg/L] of rats fed with low and high fluoride for 3 or 6 months were significantly higher than those in control group [(1.08 ± 0.15),(1.09 ± 0.17) mg/L,P ＜ 0.05].The escape latency time [(18.36 ± 2.80) s] of rats in the high fluoride group at the end of 3 months was higher than that of control group [(6.68 ± 3.01) s,P ＜ 0.05],the number of stay time in platform quadrant [(12.91 ± 3.25) s] was lower than that of control group [(19.97 ± 3.30) s,P ＜ 0.05].The escape latency time [(15.46 ± 4.56),(28.16 ± 4.00) s] of rats in low and high fluoride groups at the end of 6 months were all higher than that of control group [(6.62 ± 2.31) s,P ＜ 0.05];the number of crossing the platform and stay time in platform quadrant [(2.25 ± 1.71) times,(12.73 ± 3.55) s;(1.40 ± 1.15) times,(9.26 ± 1.72) s] of these rats were significantly lower than those of the control group [(4.00 ± 1.58) times,(19.53 ± 4.36) s,P ＜ 0.05].The expression levels of protein [(73.84 ± 9.68)％,(73.23 ± 4.51)％;(53.30 ± 17.63)％,(54.69 ± 18.71)％] and mRNA [(70.33 ± 4.89)％,(66.27 ± 3.38)％;(37.72 ± 4.89)％,(44.15 ± 1.74)％] of SIRT1 in the hippocampus and cortex of rats fed with high fluoride for 3 or 6 months were significantly lower than those in control group [(100.00 ± 13.51)％,(100.00 ± 13.60)％;(100.00 ± 15.37)％,(100.00 ± 12.19)％;(100.00 ± 2.65)％,(100.00 ± 4.34)％;(100.00 ± 3.40)％,(100.00 ± 4.52)％,P ＜ 0.05].Whereas,the decreased expression levels of protein [(77.65 ± 14.51)％,(71.51 ± 8.27)％] and mRNA [(57.78 ± 1.96)％,(63.76 ± 2.16)％] of SIRT1 in the hippocampus and cortex of rats in the low fluoride group were only observed at the end of 6 month of experiment (P ＜ 0.05).The expression of protein of SIRT1 in the hippocampus and cortex of rats in 3 or 6 months was negatively correlated with the escape latency time of rats (r=-0.598 5,-0.493 2;-0.782 6,-0.777 3,P＜ 0.05),and it was positively correlated with the number of crossing the platform (r =0.547 7,0.523 3;0.720 5,0.715 4,P ＜ 0.05).Conclusion The decrease of the ability of learning and memory in rats with chronic fluorosis may be related to the decreased expression of SIRT1 influenced by chronic fluorosis.

Objective To detect the expression of peroxisome proliferator-activated receptor γ (PPARγ) in the brains of rats with chronic fluorosis and elucidate the relationship between PPARγand oxidative stress in chronic fluorosis.Methods According to body weight (100-120 g),sixty healthy SD rats were divided into control group (less than 0.5 mg/L fluoride in drinking water),low fluoride group (5.0 mg/L fluoride in drinking water,prepared by NaF),and high fluoride group (50.0 mg/L fluoride in drinking water) via the random number table method,20 rats in each group (half male and half female).The experiment periods were 3 and 6 months,respectively.Then 24-hour urine samples of rats were collected from each group,all rats were put to death and brain tissues were taken.The fluoride contents in urine and brain tissue were measured with fluoride-ion selective electrode;the levels of PPARγ protein and mRNA in the cortex and hippocampus were determined by Western blotting and Real-time fluorescence quantitative PCR,respectively;and the activities of superoxide dismutase (SOD) and level of malondialdehyde (MDA) in serum were detected by xanthine oxidase method and thiobarbituric acid method;the correlation between PPARγ protein expression and oxidative stress was analyzed.Results After 3 and 6 months of treatment,the contents of fluoride in urine and brain in low fluoride group [(1.57 ± 0.18) mg/L,(3.43 ± 0.70) μg/g;(1.79 ± 0.17) mg/L,(7.40 ± 1.21) μg/g] were higher than those of control group [(1.11 ± 0.17) mg/L,(2.39 ± 0.50) μg/g;(1.02 ± 0.15) mg/L,(2.87 ± 0.82) μg/g,P ＜ 0.05],and the values in high fluoride group [(1.91 ± 0.23) mg/L,(6.70 ± 0.87) μg/g;(2.44 ± 0.51) mg/L,(12.10 ± 1.30) μg/g] were significantly higher than those in low fluoride group (P ＜ 0.05).In high fluoride group after 3 months of treatment,the expression of PPARγprotein [(79.00 ± 3.46)％,(80.35 ± 2.50)％] and mRNA [(79.11 ± 11.18)％,(82.10 ± 9.94)％] in hippocampus and cortex of rat brains were significantly lower than those of low fluoride group [(104.01 ± 5.77)％,(101.17 ± 6.35)％;(112.88 ± 22.15)％,(101.14 ± 8.60)％,P＜ 0.05];the expression of PPARγprotein [(64.32 ± 10.43)％,(60.20 ± 10.92)％] and mRNA [(41.03 ± 9.93)％,(52.25 ± 11.48)％] in the same brain regions of the rats after 6 months of treatment in high fluoride group were significantly lower than those of control group [(99.99 ± 11.19)％,(100.00 ± 11.30)％;(100.00 ± 10.00)％,(100.00 ± 9.00)％] and low fluoride group [(73.88 ± 3.36)％,(81.50 ± 14.90)％;(76.02 ± 8.65)％,(73.36 ± 7.43)％,P ＜ 0.05].The activities of SOD in serum in low and high fluoride groups after 6 month treatment [(37.94 ± 1.92),(35.54 ± 2.53) U/ml] were significantly lower than that of control group [(41.24 ± 0.66) U/ml,P ＜ 0.05],and the value in high fluoride group was lower than that in low fluoride group (P ＜ 0.05);serum MDA contents in high fluoride group after 3 and 6 month treatment [(8.29 ± 1.49),(11.63 ± 1.04) nmol/mg pr] were higher than those in low fluoride group [(6.39 ± 0.69),(7.50 ± 1.64) nmol/mg pr] and control group [(5.02 ± 0.71),(5.87 ± 1.03) nmol/mg pr,P ＜ 0.05].The correlation analysis results showed the levels of PPARγprotein in hippocampus and cortex of rats were negatively correlated with fluoride contents in brain tissues (3 month:r=-0.769,-0.793;6 month:r =-0.832,-0.870;P ＜ 0.05),positively correlated with SOD activities (3 month:r =0.550,0.826;6 month:r =0.822,0.896;P ＜ 0.05) and negatively correlated with MDA contents (3 month:r =-0.703,-0.609,6 month:r =-0.792,-0.657;P ＜ 0.05) in serum.Conclusions Declined expression of PPARγat protein and mRNA levels has been detected in brains of rats with chronic fluorosis,which might be related to the increase of oxidative stress.PPARγ may be involved in the occurrence of chronic fluorosis.