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    氟斑牙大鼠發(fā)生纖維瘤:內(nèi)質(zhì)網(wǎng)應(yīng)激和線粒體的激活途徑

    2021-11-13 07:26:40曾藹霖李惠如胡小艷楊敏芝母梅力劉建國(guó)吳明松
    關(guān)鍵詞:氟斑牙劉建國(guó)楊敏

    曾藹霖,李惠如,胡小艷, 楊敏芝,母梅力,陳 姚,劉建國(guó),2,吳明松,2

    (1.遵義醫(yī)科大學(xué) 口腔醫(yī)學(xué)院,貴州 遵義 563099;2.遵義醫(yī)科大學(xué) 貴州省普通高等學(xué)??谇患膊⊙芯刻厣攸c(diǎn)實(shí)驗(yàn)室,貴州 遵義 563099)

    Fluorine is a necessary trace element of organisms,and is good for health so that the fluoride is routinely added in toothpaste and public water in many countries.Nevertheless,excessive fluoride may bring about damage to animals.Its hazard is mainly in violation of teeth and bones,causing dental fluorosis and bone fluorosis,extensive distribution,and the number of patients[1].fluorosis is a kind of endemic disease throughout the world,and the dental and skeletal fluorosis are common diseases in clinic.Hence it is a controversial issue of the fluoridation.

    Currently,tumorigenesis of excessive fluoride is concerned by more and more scientists who propose that fluoride interferes with the normal biology or/and genetics of the body,and further suppose that fluoride is linked to the development of osteosarcoma[2].However,others believe that there is no evidence that higher levels of fluoride lead to a greater risk of either osteosarcoma or Ewing sarcoma[3].It is worth noting that most of the debates focused on the bone tumor.So we report here on a kind of soft tissue tumor from a rat with dental fluorosis in order to provide a case to the debate.

    Moreover,fluoride induces endoplasmic reticulum stress (ERS) and alters Ca2+signaling[4-5]and mitochondrial function[6]in enamel cells.ERS and mitochondria oxidative stress play a vital role in tumorigenesis.In the study,the transcriptional changes of ERS- and mitochondria- pathway genes were investigated in the soft tissue tumor.

    1 Materials and methods

    1.1 Animals Twenty adult male Sprague-dawley rats,aged 19 weeks,weighing (300±20)g,half male and half female,were purchased from the Animal Experimental Center of Zunyi Medical University,and housed with five rats of the same sex in a cage.Feeded with standard rat chow,the rats were kept in the (20±3)°C of ambient temperature and maintained in good ventilation and light.The animal experimental procedures for this study were reviewed and approved by the Institutional Animal Use and Care Committee of Zunyi Medical University.

    1.2 Experimental design and collection of samples After adaptive feeding under suitable environmental conditions for one week,the rats were fed with 0.15 g/L sodium fluoride solution (Sigma-Aldrich) for 14 months,then sacrificed under anesthesia,and the tumor tissues were collected for Hematoxylin and eosin staining according to a normal procedure.

    1.3 PCR procedure The tissues collected from two different parts of the tumor tissues (T) and para-cancerous tissues (P),respectively.The total RNA was isolated from the tissues and synthesized the first-strand cDNA with Prime script RT Enzyme mix (Takara,Dalian,China).The q-RT-PCR reactions were performed on a CFX Connect Real-Time System (BioRad,USA) using the SYBR Green dye method.The q-RT-PCR reactions were performed in 10 μl volumes that included the forward and reverse primers (10 μM) (seen Table 1).The PCR was done under the conditions at 95°C for 5 minutes,then 40 cycles of amplification at 95°C for 30 seconds,60°C for 30 seconds.Each gene was normalized to the internal β-Actin level.Each sample was run in triplicate to ensure quantitative accuracy,and the threshold cycle numbers (Ct) were averaged.The results were calculated using the 2-ΔCtmethod.

    Table 1 The sequence of the PCR primers

    1.4 Data analysis The levels of genes were respectively analyzed using the t-test by Graphpad Prism 8.0.Differences were considered significant ifP<0.05.

    2 Results

    The incisors of rats showed white or chalky color (Fig 1A),which was a typical characteristic of dental fluorosis.But there was just one rat with tumor among the SD rats with dental fluorosis.It was poor mental state with rough hair.At the same time,the tumor or neoplasm between the subcutaneous fat layer and the visceral fascia layer of the left lower abdomen was about 3.5 cm and 4.0 cm in size,with the intact capsule,hard texture and high mobility (Fig 1B).After incision,it was pink without necrosis (Fig 1C).The histologic section (Fig 1D) showed that the tumor was composed of a large number of fibrous cells,which were spindle cells,less cytoplasm,weakly eosinophilic,oval nucleus and deep staining.It widely invaded the sarcolemma,subserosa and further extended to the mucous membrane and serosa surface of the lamina propria.From the different sections of the slice,there were a large number of fibrous connective tissues accompanied by scattered deep-stained fibrous cells in the oval nucleus in the external area.The fibrous cells arranged in cords and the infiltration was aggravated inside the area,as well as the extensive scope of cell infiltration,several narrow cord-like gaps were running through the whole inside area.Furthermore,the neoplasm was infiltrated by dense fibrous cells,and the intercellular space could no longer be seen in most places in the core area.These pathological features suggested that it was a benign fibromalike tumor.

    Compared with para-cancerous tissues,the expression of ERS moleculesGRP78,GRP94 andCHOPup-regulated in tumor tissues (Fig 2A,B,C),suggesting that ERS was activated in tumor tissues.In the signaling pathways,the expression ofIRE1,PERK,elF2αandATF4 was increased (Fig 2D,E,F(xiàn),G,H,I),but the level ofXBP1 andATF6 was down-regulated.In the mitochondrial pathway,the levels ofcytochromeC(Cyt-C),BAXandBCL2 raised (Fig 2J,K,I).However,BCL2 increased 3-fold in tumor tissues,exceeding the elevated levels ofCyt-CandBAX.The down-regulation ofcaspase3 (Casp3) (Fig 2N) demonstrated that apoptosis level decreased significantly and reflected the high growth activity in the tumor tissue.

    The degree of dental fluorosis (A),the site (B) and size (C) of the soft tumor were shown.Histological sections were stained by hematoxylin eosin (D) from the external,inside and core tumor tissues.Figure 1 The histopathological characteristics of the tumor tissues

    The relative expression levels of genes were normalized by β-actin in tumor tissues (T) and para-cancerous tissues (PT).*P<0.05 or **P<0.01,compared with that in para-cancerous tissue.Figure 2 The relative levels of ER stress- and mitochondrial apoptosis-related genes in the tumor tissues

    3 Discussion

    It has been reported in some literatures that excessive fluorine can cause tumorigenesis.Nowadays,fluoride has been widely used in people’s daily life,biomedicine and other extensive fields.Significantly,countries use fluorinated water and toothpaste to prevent caries.In addition,18F-NaF has become one of the diagnostic criteria of bone diseases[7]or bone metastases of tumors[8],but its safety also needs more research.This study may provide a reference for it.

    Consequently,the safety of fluoride becomes more important in the society.In this study,among the twenty sodium fluoride model rats,a male rat incidentally suffered from a fibrous like tumor.However,We found only one rat with soft tissue tumor in rats with dental fluorosis and it is worth pointing out that the concentration of fluoride for these rats was much higher than that used by people to prevent caries.Therefore,more scientific evidence is needed to determine whether this tumor is the result of fluorosis.Even so,this is the first case of abdominal soft tissue tumors in dental fluorosis rats,providing reference for the relation between the tumors and excessive fluoride.

    The fluorosis process is the adaptation of rats to fluoride in the environment,so fluoride is also an environmental stress factor,which leads to apoptosis of bone cells and ameloblasts,while ERS is not only one of the mechanisms of adaptation to the environment,but also one of the essential molecular mechanisms of cell apoptosis.Therefore,we observed the changes of some transcriptional gene levels of endoplasmic reticulum stress and mitochondrial apoptosis pathway in the tumor and found that ERS and mitochondrial molecules were activated in the tumor tissues.Interestingly,among the three ERS pathways,IRE1 and PERK pathways were up-regulated,while the ATF6 pathway was down-regulated,indicating that different ERS pathways play different roles in fluoride-induced soft tissue tumors,even antagonizing each other.Taken CHOP for another example,CHOP induces cell apoptosis[9],but it was also up-regulated in the neoplasm tissue.Hence the occurrence of the tumor is the result of the comprehensive action of many factors.The unfolded protein response (UPR) is an adaptive pathway that restores cellular homeostasis after endoplasmic reticulum stress and maintains cellular homeostasis[10]by producing energy or eliminating misfolded or aggregated proteins.

    Endoplasmic reticulum stress is not only involved in the occurrence and development of tumors but also the formation of dental fluorosis.In the experimental course,the rat with fluorosis was dispirited,and their hair was rough and yellow.This kind of mental disorder and the tumor was one of the manifestations of the integrated stress response (ISR)[7,11].To maintain the normal function,the cells must synthesize the appropriate amount of protein,and the synthesized proteins should be folded and assembled correctly,located accurately and degraded in time.Once this process is abnormal,stress molecules will initiate the regulation of intracellular signal networks.ISR is a conservative response network that can respond to stress in the endoplasmic reticulum and cytoplasm[12].Unfolded protein response can recognize misfolded proteins in the endoplasmic reticulum.Heat shock response (HSR) leads to an increase of GRP78/GRP94 and promotes the correct folding and degradation of proteins.ISR can be coupled to UPR and HSR[10].ISR can lead to protein transcriptional reprogramming,which not only regulates the gene expression of endoplasmic reticulum stress pathway[12],but also regulates the BCL2 gene that promotes cell survival,promotes cell survival,inhibits apoptosis,and leads to the occurrence and development of the tumor.There was no significant change,but the apoptosis level of tumor tissue decreased significantly,reflecting the high growth activity of tumor tissues.

    To sum up,during the study of rat dental fluorosis,we found that a soft tumor developed in the abdomen of a rat dental fluorosis and its molecular mechanisms were involved in ER stress and mitochondrial pathways,which provides evidence for the possible correlation between dental fluorosis and fibroma and the tumorigenesis of sodium fluoride.However,more experimental evidence is needed.

    Dental fluorosis maybe associated with a benign fibroma of rat,and its molecular mechanisms are link to the ERS and mitochondrial- pathways,providing probable evidence for the tumorigenesis caused by sodium fluoride.

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