Objective To investigate the pathological role of endoplasmic reticulum stress in fluomsisinduced apoptosis in human hepatocellular carcinoma cell line (HepG2).Methods Under stimulation of 1,3,6,9 mmol/L concentrations of NaF in vitro for 24 h,while normal control group was cultured under normal condition,the apoptosis of HepG2 cells was measured by flow cytometry.The endoplasmic reticulum stress markers (glucose regulative proteins 78,94;GRP78,GRP94) and CCAAT/enhancer-binding protein homologous protein (CHOP) in HepG2 cells were measured at both mRNA and protein levels by real-time PCR and Western blotting,respectively.Results After treated with 0,1,3,6,9 mmol/L NaF for 24 h,the apoptosis rate of HepG2 cells was (6.25 ± 1.27)％,(13.48 ± 1.00)％,(24.08 ± 1.88)％,(30.19 ± 3.07)％ and (37.72 ± 4.43)％,respectively,and the difference was statistically significant among groups (F =65.828,P ＜ 0.01).After treated with 3 mmol/L NaF for 24 h,the mRNA level of GRP78,GRP94 and CHOP was (1 172.41 ± 459.60)％,(946.95 ± 635.85)％ and (7 846.97 ± 1 670.01)％,which was increased compared to those of the control groups [(100.00 ± 1.77)％,(100.00 ± 2.08)％,(100.00 ± 0.74)％,t =12.77,4.67,11.50,all P ＜ 0.01].Under the same condition,the protein levels of GRP78 and CHOP were (159.99 ± 67.59)％ and (155.15 ± 94.24)％,which were increased compared to those of the control groups [(100.00 ± 30.68)％,(100.00 ± 41.44)％,t =-3.27,-1.99,all P ＜ 0.05],while GRP94 protein level [(46.40 ± 41.46)％] was decreased compared to that of the control group [(100.00 ± 68.86)％,t =4.02,P ＜ 0.05].Conclusion Endoplasmic reticulum stress may be involved in NaF-induced cell death in HepG2 cells.

Objective To investigate fluoride-induced inflammation and nuclear factor-κB (NF-κB) signaling pathway in cultured human acute monocytic leukemia cells (THP-1).Methods In vitro cultured THP-1 cells were used as a model of microglia.After cultured with different concentrations of [0 (negative control group),10,50,100,500,1 000 and 5 000 μmol/L] sodium fluoride (NaF) for 48 h,the survival of cells was detected by CCK8.THP-1 cells were divided into 3 groups:control group,low dose and high dose fluoride groups according to the results of CCK8 assay,and then treated with different concentrations of sodium fluoride (0,500,5 000 μmol/L) for 48 h,concentrations of inflammatory cytokines,such as Interleukin-lβ (IL-1β) and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA) in THP-1 mononuclear cell culture medium.The protein levels of IκBα,phospho-NF-κB p65 and phospho-IκB-α were detected by Western blotting.Results THP-1 cells were treated with different concentrations of sodium fluoride (500,1 000,5 000 μ mol/L) for 48 h.Fluoride group THP-1 cell survival rate [(73.21 ± 3.67)％,(31.40 ± 4.56)％,(0.40 ± 0.24)％] was lower than that of the negative control group [(100.00 ± 0.00)％,all P ＜ 0.01].Compared to the control groups [(0.36 ± 0.07),(31.07 ± 0.81)ng/L],significant increases of the inflammatory cytokines IL-1β [(1.42 ± 0.79),(19.47 ± 2.90)ng/L] and TNF-α [(61.06 ± 2.20),(172.72 ± 2.29)ng/L] were detected in culture medium in low-fluoride and high fluoride groups,respectively.Interestingly,compared to the control groups [(100.00 ± 5.48)％,(100.00 ± 14.82)％],significant increases of phospho-NF-κB p65 [(113.71 ± 8.99)％,(134.74 ± 1.93)％] and phospho-IκB-α [(152.61 ± 14.16)％,(176.91 ± 7.95)％] were observed in both low-fluoride and high fluoride groups.Meanwhile,the protein level of IκBα in high fluoride group [(63.53 ± 9.67)％] was significantly lower than that of the control group [(100.00 ± 10.99)％,P ＜ 0.01].Furthermore,significant positive correlation was detected between increased IL-1β,TNF-α and phospho-NF-κB p65 (r =0.74,0.72,all P ＜ 0.05).Conclusions Excessive fluoride can induce microglial cells to release inflammatory cytokines and activate nuclear factor-κB signaling pathway.The release of inflammatory cytokines and activation of the signaling pathway may be one of the mechanisms of the damage of the central nervous system caused by sodium fluoride.

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 possible mechanism of endonuclease G (Endo G)-mediated non-Caspase-dependent apoptotic pathway in brain neuronal apoptosis in chronic fluorosis rats.Methods Sixty Sprague Dawley (SD) rats (half male and half female) were randomly divided into two groups:control group fed with tap water with fluoride content ＜ 0.5 mg/L and fluorine group in which sodium fluoride was added into drinking water with fluoride content of 50.0 mg/L.Both groups were fed with standard food with fluorine content ＜ 0.5 mg/kg.The experiment period was 10 months.At the end of the experiment,all the animals were sacrificed,and brain tissue was taken.Flow cytometry was used to examine apoptosis rate,immune-histochemistry was employed to detect the distribution of Endo G in brain tissue;Western blotting was used to test the protein expression of Endo G.Results Compared to the apoptosis rate of control group [(1.3 ± 0.6)％,(1.9 ± 0.3)％],the apoptosis rate in hippocampus and cortex of rats with chronic fluorosis [(2.6 ± 0.6)％,(3.1-± 0.7)％] was significantly increased (t =3.1,3.4,all P ＜ 0.05).The Endo G positive neurons and their degree of staining in CA1,CA2,CA3 and CA4 of hippocampus,frontal cortex as well as the upper layer of parietal cortex [(11.1 ± 2.2),(10.2 ± 1.9),(9.8 ± 3.1),(9.9 ± 1.6),(10.6 ± 2.9),(8.2 ± 2.4),(11.1 ± 2.8) scores] in rats with chronic fluorosis were significantly higher than those in the control group [(5.8 ± 1.8),(6.7 ± 2.6),(5.2 ± 2.4),(7.2 ± 2.1),(7.7 ± 2.6),(6.1 ± 1.9),(8.1 ± 2.6) scores,t =2.9,2.5,2.4,2.3,2.2,2.5,2.3,P ＜ 0.01 or ＜ 0.05].The protein level of Endo G in the mitochondria of rat brains with chronic fluorosis [(86.4 ± 7.2)％,(83.9 ± 6.8)％] was significantly lower than that of control group [(100.0 ± 6.1)％,(100.0 ± 5.5)％,t =2.6,2.3,all P ＜ 0.05].Meanwhile,the protein level of Endo G in the nucleus of neurons from chronic fluorosis rats [(117.5 ± 6.4)％,(115.2 ± 6.2)％] was significantly higher than that of the control [(100.0 ± 5.2)％,(100.0 ± 5.5)％,t =2.5,2.2,all P ＜ 0.05].Conclusion The high expression of Endo G and nuclear transfer are related to the neuron apoptosis in chronic fluorosis rat,which may be one of the mechanisms of brain injury of the disease.

Objective To observe the effect of treating Diabetic Peripheral Neuropathy with Acupoint Injection. Methods 65 patient with DPN were randomly recruited into a control group and a treatment group after adjusting stage. The control group was treated with acupuncture, while the treatment group was treated with the Safflower inoculation fluid acupuncture point injects. Nerve function parameter (MDNS、 NCV) and clinical symptoms were observed after 3 therapeutic courses. Results Nerve function parameter (MDNS、 NCV) and clinical symptoms in the treatment group were apparently improved after treatment (P<0.05) . Conclusion Acupuncture point injected with Safflower inoculation fluid may improve clinical symptoms and nerve functions of DPN patients.

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 explore the role of apoptosis-inducing factors (AIF) mediated non-apoptosis classic pathway involved in neuron apoptosis of rats with chronic fluorosis.Methods Sixty Sprague Dawley (SD) rats (body weight 100-120 g) were divided into two groups (30 rats in each group,half male and half female) by random number table according to body weight.Control group was fed with tap water with fluoride content ＜ 0.5 mg/L and fluorine group was fed with water with fluoride content of 50.0 mg/L.Both groups were fed with standard food with fluorine content ＜ 0.5 mg/kg.After 10 months,all the animals were sacrificed though heart perfusion using phosphate buffer,and brain tissue was taken.Immunohistochemical method was employed to detect the distribution of AIF in brain tissue.Western blotting was used to test the protein expression of AIF,cl-caspase-3 and cl-caspase-9.Flow cytometry was used to examine apoptosis rate.Results The AIF positive distribution and degree of staining in the neurons of hippocampal CA1,CA2 and CA3,as well as the parietal cortex (7.50 ± 2.17,9.00 ± 1.63,8.00 ±0.82,10.24 ± 1.80) in rats with chronic fluorosis were significantly higher than those of the control group (5.18 ±1.66,6.27 ± 1.42,6.36 ± 1.96,6.96 ± 2.62,t =2.76,4.09,2.45,5.77,all P ＜ 0.05).The AIF protein expression of neuronal mitochondria in the cerebral tissue of rats with chronic fluorosis [(89.46 ± 8.47)％] was significantly lower than that of the control group [(100.00 ± 7.12)％,t =3.16,P ＜ 0.01],while the AIF protein expression of the neuronal nucleus [(112.80 ± 7.10)％] was significantly higher than that of the control group [(100.00 ± 8.20)％,t =3.75,P ＜ 0.01];cl-caspase-3 and cl-caspase-9 protein expression in the neurons of hippocampus and cortex from the rats with chronic fluorosis [(132.14 ± 18.66)％,(107.31 ± 2.58)％,(121.33 ± 14.86)％,(112.97 ± 7.97)％]were significantly higher than those of the control group [(100.00 ± 11.99)％,(100.00 ± 3.74)％,(100.00 ± 16.87)％,(100.00 ± 8.04)％,t =3.55,3.94,2.32,2.81,P ＜ 0.01 or ＜ 0.05].As compared with those of the control group [(1.28 ± 0.59)％,(1.88 ± 0.25)％],the apoptosis rates in hippocampus and cortex of the rats with chronic fluorosis [(2.55 ± 0.58)％,(3.05 ± 0.65)％] were significantly increased (t =3.08,3.40,all P ＜ 0.05).Conclusion Both of AIF-mediated caspase-independent apoptosis and the classic caspase-dependent apoptosis pathways have participated in neuron apoptosis in rat induced by chronic fluorosis,which may be one of the mechanisms of brain damage of the disease.

Objective To investigate the association between HLA-DP gene polymorphism and the susceptibility to hepatitis B virus (HBV) infection in people in Buyi,Miao and Shui ethnic groups in Guizhou.Methods A total of 256 patients with HBV infection,142 HBV self-cleared patients and 135 controls were recruited from 3 ethnic groups for this case-control study.Genotypes of rs3077 and rs9277535 were determined by using real-time PCR with a TaqMan-MGB probe.Results Compared with healthy subjects and self-cleared subjects,the allele distribution of rs9277535 was significantly associated with chronic HBV infection (P＜0.05),no significant differences in rs3077 allele and genotype distributions were found among 3 groups (P＞0.05).Compared with the rs9277535 GG genotype,the AA and AG genotypes were protective factors against HBV infection in dominant model after adjustment for age and sex (OR=0.645,95％CI:0.421-0.988),and no significant difference in rs3077 locus was found in the same analysis (P＞0.05).For men,the rs3077 locus CC and CT genotypes were also protective factors against HBV infection (OR=0.493,95％CI:0.266-0.916),and there was no significant difference in rs9277535 locus compared the genotype distributions among 3 groups in dominant mode.Comparison of allele and genotype distribution in 3 ethnic groups showed that HLA-DP gene rs3077 genotype distributions had significant difference between the HBV infection group and the healthy control group in Buyi ethnic group (x2=6.726,P=0.036).Conclusions The presence of rs9277535A allele at HLA-DP gene polymorphisms might decrease the risk for HBV infection,the rs3077 C allele at HLA-DP gene polymorphisms might also confer protective effect against HBV infection in women.No significant correlation between HLA-DP gene rs3077 and rs9277535 locus polymorphism and HBV clearance was found in this study.The HLA-DP gene rs3077 genotype distribution of HBV-infected patients had significant differences among different ethnic groups.

Objective To explore the availability and safety of ileal ureter replacement combined with ileal augmentation cystoplasty in treating ureteral stenosis with contracted bladder.Methods From August 2015 to November 2018,three patients who underwent ileal ureter replacement combined with augmentation cystoplasty were treated with ileal ureter replacement combined with augmentation cystoplasty.There were 1 male and 2 females with the age ranging from 34 to 55 years (mean 39 years).Two patients suffered from left ureter stenosis,and one patient had stenosis on the both sides.The length of the ureter stenosis ranged from 6 to 18 cm (mean 9.8 cm).The preoperative bladder capacity ranged from 60 to 150 ml (mean 103.3 ml).In the surgery,part of ileum was used to replace the ureter,and the distal intestine was made into U-shape to enlarge the bladder.Results All operation were completed successfully.The operation time ranged from 220 to 400 min (mean 303.0 min),and the blood loss ranged from 150 to 500 ml (mean 283.3 ml).Laparoscopic surgery was performed in 1 case and open surgery in 2 cases.Three weeks after the surgery,the bladder volume underwent cystography ranged from 300 to 400 ml (mean 360.0 ml).Three months after the surgery,the postvoid residual urine volume ranged from 20 to 50 ml (mean 33.3 ml).Postoperative frequency and urgency of urine were completely relieved in 1 case,alleviated in 2 cases.Flank pain was completely relieved in 1 case,and alleviated in 2 cases postoperatively.The serum creatinine of 2 patients mildly increased after the surgery,while 1 patient remained stable.For complications,1 patient had urinary infection,and 1 patient suffered from metabolic acidosis.Conclusions Ileal ureteral replacement combined with augmentation cystoplasty can be the choice of treatment for long segment ureteral stenosis and enlarge the bladder simultaneously.The postoperative complications and the kidney functions should be regularly followed up.