Expression Of The Autophagy Related Factors Beclin1,lc3and P62 In The Damage Of Chondrocytes In The Chronic Fluorosis Rats

Objective: To reveal autophagy in chronic fluorosis function and mechanism in vivo and in vitro through investigating the expression of protein and mRNA of autophagy related factor Beclin1,LC3 in cartilage and chondrocytes.Methods:(1)36 healthy adult SD(Sprague-Dawley)rats were randomly divided into3 groups,12 rats in each group.The control rats were provided with tap water in which the fluorine concentration was <1mg/L,while low fluoride group and high fluoride group were provided fluoridation by drinking water,which the fluorine concentration was 5 mg/L and 50 mg/L,respectively.After 6 months of experiment treatment,the rats were euthanized using bloodletting in the femoral artery,the fluorine contents in urine and bone were detected with the fluorine-ion electrode method.Histological changes in cartilage tissues were stained by Hematoxylin &Eeosin(H&E)and observed under light microscopy.The protein levels of components expression of Beclin1,LC3,p62,Bcl-2 in cartilage of experimental animals were determined by immunohistochemistry(IHC)and Western Blot.(2)Primary cartilage cells were obtained from articular cartilage of neonatal Sprague-Dawley rats using a mechanical enzymatic digestion method.The primary chondrocytes were identified with toluidine blue.The cell viability treated with0(control),5,10,20,40 mg/L of fluoride after cultured for 24,48,72 h was tested using MTT assay.The protein and m RNA levels of Beclin1,LC3 were tested by western blot analysis and RT-PCR,respectively.The protein of Beclin1,LC3 expressions in the endoplasmic reticulum of cartilage cell were tested under the laserconfocal microscopy.Results: 1.The different degree of dental fluorosis was observed in the rats treated by fluoride,compared with control group.The fluorine contents in the urine and bone in low and high-dose fluoride groups(2.72±0.11,4.43±0.14;237.67±8.33,270.25±9.83)were higher than those of control group(1.83±0.10;182.58±12.02)(P<0.05).2.The cartilage cells columns obviously thinner and disordered arrangement were also observed with the increase of dye fluorine dose in the group treated by fluorine agent,compared with control group.3.The results of immunohistochemistry showed that the protein expressions in low and high-dose fluoride groups of Beclin1(67.75±8.67,85.75±12.29),LC3(68.17±12.18,86.58±11.07),p62(55.92±8.22,39.58±7.07)and Bcl-2(78.00±6.22,59.17±5.25),compared with control group the Beclin1(52.92±8.63),LC3(50.92±9.36),p62(71.42±8.72),Bcl-2(101.58±6.73)(P < 0.05).4.The Wb showed that,the protein levels of Beclin1,LC3,p62,Bcl-2 in low and high-dose fluoride groups,Beclin1(0.50±0.06,0.63±0.06),LC3(0.56±0.08,0.74±0.08),p62(0.59±0.06,0.27±0.03),Bcl-2(0.61±0.08,0.37±0.07),compared with control group the Beclin1(0.40±0.08),LC3(0.42±0.06)were significantly increased,compared with control group the p62(0.79±0.09)and Bcl-2(1.15±0.10)were significantly decreased(P<0.05).5.The primary chondrocytes were successful obtained.In 24,48,72 h the proliferation rates in 5 mg/L fluoride treatment groups were(1.15±0.07,1.19±0.04,1.07±0.22)and in10mg/L fluoride treatment groups were(1.16±0.13,1.22±0.06,1.07±0.18)increased compared with control group[(1.02±0.04,1.01±0.11,1.00±0.05)].In 24 h,48h the proliferation rates were obviously increases,but 5mg/L group compared with 10mg/L group proliferation rate had no definite trend in each time period.6.In the fluoride(10mg/L,20mg/L,40mg/L)groups at 48 h the protein expressions of Beclin1(0.64±0.10,0.77±0.14,1.88±0.21),LC3(0.72±0.11,1.36±0.19,2.02±0.26),and the mRNA expressions of Beclin1[(0.52±0.05,0.67±0.12,0.99±0.13)] and LC3[(0.72±0.12,0.94±0.07,1.19±0.13)] were increased respectively,compared with control group protein of Beclin1(0.32±0.10),LC3(0.43±0.07)and the mRNA of Beclin1(0.22±0.03),LC3(0.33±0.06)(P<0.05).7.The protein in endoplasmic reticulum in cartilage cells expressions of Beclin1(238.33±16.01,350.67±27.39,455.67±29.54),LC3(440.67± 36.07,439.33±35.02,499.67±24.17)were increased,compared with control group Beclin1 protein(185.67±20.82)and LC3 protein(217.67±25.01)(P<0.05).The average optical densities of endoplasmic reticulum,compared with control group(273.40±26.42)in 10mg/L,20mg/L,40mg/L groups(267.93±24.10;275.13±26.66;293.17±25.43)had no statistical significance(P>0.05).Conclusion: The proliferation and apoptosis of cartilage cell of rats are promoted in different concentration fluoride.The expressions of Beclin1,LC3,p62,Bcl-2 protein in cartilage related factors of autophagy and apoptosis of rats caused by excessive fluorine are abnormal.The factors related autophagy could be activated by endoplasmic reticulum stress through excessive fluoride,then it influences to the endoplasmic reticulum dysfunction of rat's chondrocytes treated by excessive fluorine.The autophagy in the endoplasmic reticulum may be one of the mechanisms of the articular cartilage damage caused by chronic fluorosis.