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Objective:To explore whether chronic fluorosis can cause liver fibrosis in rats by observing expression changes in type Ⅰcollagen (Col-Ⅰ), type Ⅲ collagen (Col-Ⅲ) and alpha smooth actin (α-SMA) in the liver tissue of chronic fluorosis rats.Methods:According to body weight (90 - 100 g), forty-eight SD rats were randomly divided into control group (drinking water fluoride ion concentration < 0.5 mg/L), low, medium and high concentration fluoride groups (drinking water fluoride ion concentration of 5.0, 50.0 and 100.0 mg/L), with 12 rats in each group (half male and half female), and fed for 6 months. Fluoride ion selective electrode method was used to detect bone fluoride and urinary fluoride levels; hematoxylin-eosin staining (HE staining) and Masson staining were used to observe the pathological and morphological changes and the collagen deposition of liver tissue; quantitative real-time polymerase chain reaction and immunohistochemical staining were used to observe Col-Ⅰ, Col-Ⅲ and α-SMA mRNA and protein expressions.Results:There was significant difference in bone fluoride and urine fluoride between the 4 groups [bone fluoride: (92.52 ± 5.64), (112.21 ± 11.86), (142.99 ± 7.87), (235.63 ± 11.55) mg/kg; urinary fluoride: (5.47 ± 0.88), (17.78 ± 1.48), (54.16 ± 5.96), (121.11 ± 6.32) mg/L, P < 0.001]. Under light microscope, with the increase of fluoride concentration, the degree of hepatic cell edema was aggravated, and the deposition of collagen fiber around the central vein and the portal area increased significantly. The mRNA expression level of Col-Ⅰ in low, medium and high concentration fluoride groups (1.20 ± 0.09, 1.80 ± 0.08, 1.58 ± 0.06) was significantly higher than that in control group (1.00 ± 0.00, P < 0.05); Col-Ⅲ and α-SMA mRNA expression levels in medium and high concentration fluoride groups (Col-Ⅲ: 1.15 ± 0.14, 1.64 ± 0.24; α-SMA: 1.69 ± 0.02, 2.34 ± 0.06) were significantly higher than those of low concentration fluoride group (Col-Ⅲ: 0.59 ± 0.17; α-SMA: 0.80 ± 0.13, P < 0.05). With the increase of fluoride concentration, the liver tissue Col-Ⅰ(0.00 ± 0.00, 0.03 ± 0.01, 0.08 ± 0.01, 0.13 ± 0.02), Col-Ⅲ (17 803.05 ± 3 221.16, 47 523.15 ± 3 490.10, 127 786.35 ± 13 008.86, 237 233.03 ± 47 614.63) and α-SMA (516.83 ± 181.18, 2 885.03 ± 864.92, 11 186.94 ± 2 394.08, 37 182.43 ± 12 390.59) protein levels were also increased significantly ( P < 0.05). Conclusion:Long-term excessive intake of fluorine may cause the production of collagen fibers around the central vein and the portal area of the liver in rats to increase, and then lead to the formation of liver fibrosis.
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Objective:To observe the protein and mRNA expressions of microtubule-associated protein 1 light chain 3 (LC3)B, P62 and Beclin1 in the liver of rats with chronic fluorosis, and to explore the role of autophagy in pathogenesis of liver injury induced by fluorosis.Methods:Using a group design, 54 SD rats were divided into 9 groups according to their weight (100 - 120 g) using a random number table method, each group with 6 rats, half male and half female. They were control group (NC group), low fluoride group (LF group), high fluoride group (HF group), NC + rapamycin (RAP) group, LF + RAP group, HF + RAP group, NC + chloroquine (CQ) group, LF + CQ group, and HF + CQ group. The NC group drank tap water (fluoride concentration was 0.5 mg/L), LF group drank fluoride water (fluoride concentration was 5.0 mg/L), HF group drank fluoride water (fluoride concentration was 50.0 mg/L); NC + RAP group, LF + RAP group and HF + RAP group were fed with corresponding drinking water, respectively, for 3 months, and then RAP (1.5 mg/kg) was intraperitoneally administered for 10 d; NC + CQ group, LF + CQ group and HF + CQ group were fed with corresponding drinking water, respectively, for 3 months, and then CQ (60 mg/kg) was intraperitoneally administered for 10 d. Bone and 24-hour urine samples of rats in each group were collected to detect the contents of bone fluoride and urine fluoride; liver histomorphological changes were observed through hematoxylineosin staining; protein and mRNA expressions of LC3B, P62 and Beclin1 in liver were detected by immunohistochemistry and real-time fluorescence quantitative PCR, respectively.Results:Compared with the NC group [(0.03 ± 0.00) mg/kg, (0.34 ± 0.08) mg/L], the contents of bone fluoride [(3.86 ± 0.08) mg/kg] and urine fluoride [(1.11 ± 0.16) mg/L] in HF group were higher ( P < 0.05). In the NC group, the lobule structure of liver tissue was clear, the hepatic cords were arranged in order, and the cell structure was normal. There were different degrees of hepatocyte edema in LF and HF groups. After intraperitoneal injection of RAP, compared with the corresponding fluoride group, the morphology of hepatocytes did not change significantly. After intraperitoneal injection of CQ, compared with the corresponding fluoride group, the liver cells showed obvious edema, and the degree of edema aggravated with the increase of fluoride concentration. Compared with the NC group, the protein expressions of LC3B and Beclin1 in HF group were higher ( P < 0.05), and the protein expression of P62 was lower ( P < 0.05). After intraperitoneal injection of RAP, the protein expressions of LC3B and P62 in LF + RAP group was lower than that in LF group ( P < 0.05); Compared with HF group, the protein expressions of LC3B and Beclin1 in HF + RAP group were lower ( P < 0.05). After intraperitoneal injection of CQ, protein expression of P62 in LF + CQ group was higher than that in LF group ( P < 0.05); Compared with HF group, protein expression of P62 in HF + CQ group was higher ( P < 0.05). Conclusions:Early (3 month) fluoride intake could promote autophagy and induce edema of hepatocytes in rats, and RAP had similar effects. CQ may induce liver injury by inhibiting autophagy of hepatocytes.
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Objective@#To investigate the change and association of glioma-associated oncogene homolog 1 (Gli1) and β-catenin on bone formation in rats with chronic fluorosis which were inhibited by cyclopamine (Cycl).@*Methods@#Forty-eight Sprague-Dawley rats were evenly divided to four groups, including control, F, F+Cycl and F+DMSO groups. The control group were fed with tap water (NaF<1 ppm). The F, F+Cycl and F+DMSO groups were exposed to NaF (50 ppm) in drinking water as the chronic fluorosis model. Then the rats in F+Cycl or F+DMSO groups were injected by Cycl or DMSO after 6 months, respectively. Urine fluoride concentration was detected using fluorine ion selective electrode. The enzyme-linked immunosorbent assay (ELISA) was used to detect bone alkaline phosphatase (BALP). Bone tissues were stained with hematoxylin-eosin. The mRNA and protein expression of Gli1 and β-catenin in bone tissue were detected using real-time PCR, immunohistochemistry and Western blot.@*Results@#Compared with the controls, the urine fluoride concentration and the width and volume of bone trabeculae were increased in the F, F+Cycl and F+DMSO groups, but no statistical difference among the 3 fluorosis groups. The concentration of BALP was increased in the F group and decreased in F+Cycl group (P<0.05). The expression of Gli1 and β-catenin mRNA and protein was higher in the F and F+Cycl groups than controls, but lower in the F+Cycl group than in the F group. There was positive correlation between the expression of Gli1 and β-catenin (r=0.476, P<0.05). The expression of Gli1 and β-catenin was also associated with BALP concentration and volume of bone trabeculae, respectively (r1=0.457, r2=0.466, r3=0.581, r4=0.554, respectively, P<0.05 for all).@*Conclusions@#The expression of Gli1 can be inhibited by Cycl. It may be involved in the bone formation of rats with chronic fluorosis. It may also affect the expression of β-catenin, which is an osteogenesis factor.
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Objective:To investigate the expression changes of Hedgehog related factors (Ihh, Shh and Smo) in bone of rats with chronic fluorosis, and the significance.Methods:Thirty-six healthy SD rats were divided to three groups with the method of random digits table by body weight (100 - 120 g), 12 rats in each group, half male and half female. The rats of control were fed with tap water (NaF < 1 mg/L), and the experimental rats were exposed to NaF (low dose fluoride group: 5 mg/L, high dose fluoride group: 50 mg/L) added to the drinking water to establish the chronic fluorosis model. After the rats were raised for six months, 24-hour urine samples were collected and the femoral metaphysis of the rats was taken. Urine fluoride and bone fluoride were detected by fluorin ion selective electrode method. Bone tissues were stained with hematoxylin-eosin and observed under light microscope. The content of bone alkalinephosphatase (BALP) in rats' serum was detected by enzyme-linked immunosorbent assay (ELISA). The expressions of Ihh, Shh and Smo mRNA and protein in bone were detected by Real-time PCR and immunohistochemistry (IHC).Results:The contents of urine fluoride, bone fluoride and serum BALP were increased gradually in the control, low and high doses fluoride groups [urine fluoride: (1.37 ± 0.44), (5.96 ± 0.56), (7.60 ± 0.61) mg/L; bone fluoride: (306.04 ± 12.58), (652.91 ± 51.83), (1 094.11 ± 126.34) mg/kg; BALP: (27.78 ± 4.09), (46.59 ± 5.75), (57.45 ± 3.99) U/L, P < 0.05]. It could observed that bone sclerosis by light microscope in low and high doses fluoride groups. The expressions of Ihh, Shh and Smo mRNA in high dose fluoride group (1.39 ± 0.36, 0.56 ± 0.23, 0.40 ± 0.15) were higher than those of the control and low dose fluoride groups (0.73 ± 0.19, 0.92 ± 0.34; 0.19 ± 0.04, 0.36 ± 0.16; 0.14 ± 0.04, 0.24 ± 0.13; P < 0.05). The expression of Shh mRNA in low dose fluoride group was higher than that of the control group ( P < 0.05). The expressions of Ihh and Smo protein in high dose fluoride group (138.89 ± 3.72, 149.29 ± 7.63) were higher than those of the control and the low dose fluoride groups (127.39 ± 2.69, 134.81 ± 3.53; 129.64 ± 12.62, 139.07 ± 9.30), and the low dose fluoride group were higher than those of the control group ( P < 0.05). The expression of Shh protein in high dose fluoride group (141.26 ± 7.49) was higher than that of the control group (130.96 ± 11.10, P < 0.05). Conclusion:The expression of Hedgehog signaling pathway related factors in bone of rats with chronic fluorosis is changed, which indicates that bone formation can be affected by activation of Hedgehog signaling pathway induced by fluoride.
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AIM:To investigate the effect of piceatannol on the viability, and the abilities of migration and invasion in the prostate cancer cells.METHODS:DU145 cells were treated with piceatannol at different doses (0, 5, 10, 20, 40 and 80 μmol/L) for different time (12, 24, 36 and 48 h) as indicated.The cell viability was assessed by CCK-8 assay.The migration and invasion abilities of the cells were analyzed by wound healing assay and Transwell assay, respectively.The protein levels of p-JAK2 and p-STAT3 were detected by Western blot.RESULTS:Piceatannol dose-dependently decreased the cell viability.After treatment with piceatannol, the abilities of migration and invasion of the cells were significantly inhibited.Moreover, treatment with piceatannol resulted in marked decreases in the protein levels of p-JAK2 and p-STAT3.CONCLUSION:Piceatannol inhibits the viability, migration and invasion of the prostate cancer cells via regulating the JAK2/STAT3 signaling pathway.
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Objective To study the proliferation and apoptosis and investigate the expression of Indian hedgehog (Ihh),parathyroid hormone-related peptide (PTHrp),smoothened (Smo) protein and mRNA in the cultured rat primary chondrocytes exposed to different doses of NaF.Methods The third generation articular chondrocytes of neonate rat were cultured in vitro and treated with 0 (control),5,10,20 and 40 mg/L of fluoride.The proliferation activities of cells at different times (24,48 and 72 h) were tested by Thiazolyl Blue Tetrazolium Bromide (MTT).The apoptosis rate was determined by flow cytometry.The expressions of protein and mRNA of Ihh,Smo and PTHrp at 48 h were determined by Western blotting and semi-quantitative RT-PCR,respectively.Results After exposed to 5 mg/L of fluoride for 24,48 and 72 h,the proliferation rates were significantly increased [(1.17 ± 0.07)％,(1.20 ±0.06)％,(1.16 ± 0.08)％] compared with those of control group [(1.10 ± 0.08)％,(1.13 ± 0.08)％,(1.15 ± 0.08)％],but the proliferation activity at 48 and 72 h in 40 mg/L group [(0.72 ± 0.11)％,(0.68 ± 0.04)％] was significantly lower than those in control group (all P ＜ 0.05).Compared with the control group,apoptosis rate of cartilage cell in fluoride treatment group increased gradually [(1.47 ± 0.05)％,(19.87 ± 3.03)％,(25.30 ± 1.28)％,(45.73 ± 4.63)％,F =123.328,P ＜ 0.01].Western blot analysis and RT-PCR results showed that the Ihh,PTHrp,Smo mRNA and protein expression increased in the fluoride groups at 48 h (Ihh protein:0.77 ± 0.08 vs.0.98 ±-0.07,1.23 ± 0.06,1.37 ±0.07,1.34 ± 0.07;PTHrp protein:0.68 ± 0.04 vs.0.89 ± 0.05,0.83 ± 0.05,1.29 ± 0.05,1.16 ± 0.08;Smo protein:0.37 ± 0.01 vs.0.64 ± 0.06,0.67 ± 0.03,0.96 ± 0.06,0.69 ± 0.06;Ihh mRNA:0.77 ± 0.05 vs.0.98 ± 0.05,1.09 ±0.05,1.27 ± 0.03,1.46 ± 0.06;PTHrp mRNA:0.67 ± 0.07 vs.0.97 ± 0.05,1.07 ± 0.08,1.37 ± 0.05,1.45 ± 0.05;Smo mRNA:0.45 ± 0.03 vs.0.63 ±-0.04,0.71 ± 0.05,0.81 ± 0.01,1.00 ± 0.02,all P ＜ 0.05).Conclusions Low doses of fluoride can promote the proliferation of chondrocytes cultured in vitro,and high doses of fluoride can promote the apoptosis of chondrocytes cultured in vitro.The expression of Ihh signaling pathway RNAs and proteins of the cartilage cells are increased following increased levels of fluoride.The results suggest that fluorine has activated the Ihh signaling pathway in chondrocytes and promoted the proliferation and apoptosis processes which might be involved in chondrocytes injury.
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Objective To investigate the effects of silencing Smo gene on proliferation and apoptosis of rat prima-ry chondrocyte in vitro.Methods The primary chondrocyte was obtained by mechanical-enzyme digestion and identified by Immunohistochemical cells ( ColⅡ) .The animals were divided into control group , control siRNA group and Smo siRNA 1 ~3 group.The siRNA was transfected into chondrocytes by lentivirus vector .After 72 h, the cell viability was detected by MTT, Smo expression was detected by RT-PCR and Western blot, and the apoptosis of chondrocyte was assessed by flow cytometry .Results All types of siRNA were transfected into primary chondrocyte by vectors, the Smo siRNA 1 ~3 may inhibit the expression of Smo mRNA and protein in chondrocytes, and Smo siRNA2 had the highest silencing rate ( the expressions of Smo mRNA and protein were 0.19 ±0.03 and 0.39 ±0.07 ) .The cell viability in Smo siRNA2 group was lowest ( 77.38% ±7.19%) , while the apoptosis rate of Smo siRNA2 was highest ( 21.43%±2.97%) .Conclusions Silencing Smo gene in primary chondrocytes may inhibit proliferation and promote apoptosis , Smo may have a protecting role from apop-tosis of the chondrocyte.
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<p><b>OBJECTIVE</b>To explore the impact of glycogen synthase kinase-3β (GSK-3β) on Wnt and NF-κB pathways in a rat model of diabetic nephropathy (DN).</p><p><b>METHODS</b>SD rats were randomly divided into normal control group (NC), DN model group (DM) and GSK-3β inhibitor group (DI). Blood glucose and 24-hour urine protein were monitored in three groups. Renal tissue samples were stained by HE. The expression of GSK-3β and NF-κB proteins was studied by immunohistochemistry. GSK-3β and NF-κB mRNAs were detected by RT-qPCR.</p><p><b>RESULTS</b>Ten weeks after STZ injection, the level of blood glucose increased significantly in DM group [(23.2±5.4) mmol/L] and DI group [(25.0±4.0) mmol/L], compared with NC group, and the level of 24-hour urinary protein increased significantly in DM group [(185.2±35.6) g/24 h] and DI group [(179.6±44.7) g/24 h], compared with NC group. Two weeks after LiCl injection, the level of blood glucose and 24-hour urinary protein decreased in DI group (17.6±2.1) mmol/L, (106.9±30.0) g/24 h], compared with DM Group. Compared with NC group, pathological changes of the kidney of DM group aggravated along with increased mRNA and protein expression of GSK-3β and NF-κB. But the pathological changes of the kidney in DI group alleviated along with declined mRNA and protein expression of GSK-3β and NF-κB as compared with DM group (all P<0.05).</p><p><b>CONCLUSIONS</b>NF-κB protein expression positively correlates with the GSK3β expression. Wnt and NF-κB signal pathways play an important role in the development of diabetic nephropathy.</p>
Subject(s)
Animals , Rats , Diabetic Nephropathies , Metabolism , Disease Models, Animal , Glycogen Synthase Kinase 3 , Metabolism , Glycogen Synthase Kinase 3 beta , Kidney , Pathology , NF-kappa B , Metabolism , Rats, Sprague-Dawley , Signal Transduction , Wnt Signaling PathwayABSTRACT
<p><b>OBJECTIVE</b>To investigate the expression of sonic hedgehog (Shh) signaling pathway in liver fluorosis and to explore related mechanism.</p><p><b>METHODS</b>To establish animal model, 48 normal SD rats (aged 4-5 weeks) were randomly divided into 4 groups (12 each): control group, fluoriosis group, blocking group and blocking control group. After 6 months, the blocking group and blocking control group were injected intraperitoneally once every 2 days for 3 times with 10 mg/kg cyclopamine or dimethysulfoxide, respectively. Rats were sacrificed at the end of the experiment and the fluoride content in urine and liver function was determined. The expression of Shh and Gli1 protein and mRNA in hepatocytes was detected by immunohistochemistry and real-time fluorescence quantitative PCR, respectively.</p><p><b>RESULTS</b>The fluoride contents in the urine and the incidence of dental fluorosis increased in the fluoride and blocking control groups as compared with those in the control group, but decreased in the blocking group compared with those of the fluoride and blocking control group. Compared with the control group, the titers of aspartate transaminase (AST) and alanine transaminase (ALT) significantly increased, while the activity of total protein and albumin decreased in the fluoride and blocking control groups. Compared with the fluoride and blocking control groups, the activity of the ALT slightly declined and the AST, total protein and albumin slightly increased in the blocking group. Histologically, the cells were disorganized and swollen with cytoplasmic clearing (balloon cells), compared with the control group. The expression of Shh and Gli1 significantly increased in all but the control group. Compared with the fluoride and blocking control groups, the expression of Shh and Gli1 declined in the blocking group.</p><p><b>CONCLUSIONS</b>The overexpression and cyclopamine inhibition of the Shh signaling pathway are closely related to the content of fluoride in the liver. The Shh signaling pathway plays an important role in the pathogenesis of liver injury caused by fluorosis, suggesting a preventive and therapeutic target of the disease.</p>
Subject(s)
Animals , Rats , Alanine Transaminase , Aspartate Aminotransferases , Dimethyl Sulfoxide , Pharmacology , Disease Models, Animal , Fluoride Poisoning , Drug Therapy , Metabolism , Fluorosis, Dental , Diagnosis , Hedgehog Proteins , Metabolism , Hepatocytes , Metabolism , Kruppel-Like Transcription Factors , Metabolism , Liver , Metabolism , Liver Diseases , Drug Therapy , Metabolism , RNA, Messenger , Random Allocation , Rats, Sprague-Dawley , Signal Transduction , Veratrum Alkaloids , Pharmacology , Zinc Finger Protein GLI1ABSTRACT
Objective To investigate the significance of osteo-immunology related factor transforming growth factor-β1 (TGF-β1) and interleukin 6 (IL-6) in bone of rats with chronic fluorosis.Methods Thirty-six healthy SD rats were divided to three groups according to body weight with the method of random digits table.The rats of control were fed with tap water(NaF ＜ 1 mg/L) and the experimental rats were exposed to NaF (lower dose group:5 mg/L,higher dose group:50 rmg/L) added to the drinking water to establish the chronic fluorosis model.All rats were killed on the six months and the metaphysic of femoral was collected.Bone fluorine was detected by ashing-fluorin ion selective electrode method.Bone tissues were stained with hematoxylin-eosin and observed under optical microscope.The content of bone alkaline phosphatase (BALP) in rat serum was detected by enzyme-linked immunosorbent assay(ELISA).The expressions of TGF-β1 and IL-6 mRNA and protein in bone were detected by in situ hybridization (ISH) and immunohistochemistry (IHC).Results The contents of bone fluorine were increased gradually in the control,the lower and higher doses fluoride groups[(306.04 ± 12.57),(652.91 ± 51.83),(1 094.11 ± 91.41)mg/kg,F =31.14,P ＜ 0.05].Bone sclerosis could be observed under optical microscope in lower and higher dose groups.The content of BALP in serum increased with the dose of fluoride gradually in the control,the lower and higher doses fluoride groups[(27.78 ± 4.09),(46.59 ± 5.75),(57.45 ± 3.99)U/L],expressions of mRNA (111.84 ± 4.62,123.86 ± 7.46,140.83 ± 5.21) and protein (118.60 ± 7.09,133.17 ± 7.33,145.67 ± 9.61) of TGF-β1 were both increased(F =30.29,73,28,33.65,all P ＜ 0.05).The expressions of mRNA(117.78 ± 7.01,119.90 ± 5.10) and protein(122.79 ± 6.49,123.81 ± 7.99) of IL-6 were both higher than those of the control (106.49 ± 6.76,112.11 ± 5.80,F =15.47、10.83,all P ＜ 0.05).Conclusion The expressions of osteo-immunology related factor TGF-β1 and IL-6 in bone of rats with chronic fluorosis have changed,which indicates that fluoride can impact the increased bone formation by regulating the micro environment of bone.