Study On Changes Of Serum CRP, C3, C4 And Lc Levels In Sub-chronic Overdose Fluoride-exposed Rats

Endemic fluorosis is one of the most serious endemic diseases. Its main clinical feature is skeletal fluorosis which is hard to detect. Advanced stage of skeletal fluorosis is osteoarthritis. It is reported that the osteoarthritis is similar to the degenerative arthritis clinically.The mechanism of the endemic fluorosis is related to apoptosis and oxidative damage. Oxidative stress is the essential pilot process of inflammation. C-reactive protein(CRP) is one of inflammation factors. It is the most sensitive inflammation biomarker and generated by hepatic cells. Lymphocyte is one of specific immunity leucocytes, which percentage among the leucocytes is constant. However, the percentage will be raised when chronic inflammation is occurred. Complement is a type of globin referred to immunization in body fluid. It is one of the most important factors among nonspecific immunity. Complement C3(C3) is the center part in the classical pathway and alternative pathway, which is less sensitive than complement C4(C4). Along with symptoms of inflammation, C3 and C4 will be changed together. Studies related to CRP, Lc, C3 and C4 were focused on acute infection and chronic inflammation. There were no related reports seen in endemic fluorosis field.As one of the most serious endemic diseases, endemic fluorosis caused stiffness of joint and disorder of movement, and even palsy. However, it has not effective therapy for endemic fluorosis. The therapy for inflammation is effective for the practice and is identificated. Hence, the present study was designed to illustrate the change of CRP, Lc, C3 and C4 occurring in the rats exposed to overdose fluoride and to provide some basic data for the treatment of fluorosis. Materials and Methods一,Animal grouping42 Wistar rats of both sexes, one week after ablactation(80±10g body weight) purchased from the laboratory animal department of China Medical University, were divided randomly into 3 groups according to weight and were individually housed and fed normal diets. Experiment cycle was 3 months. Grouping methods wrer as follows;Control group: 0.31 mgF~-/LLow-dose group: 100 mgF~-/LHigh-dose group: 200 mgF~-/L二,Reagents and equipmentsNaF; chloral hydrate; paraformaldehyde; absolute alcohol; haematoxylin; eosin; Beckman Coulter Immage integtation reagent: dilution, cleansing solution, menolytic agent and scattered radiation.Electronic balance; F ion selective electrode; automatic weighting scale; stainless steel metabolic cage;-20℃refrigerator;-70℃refrigerator; optical microscope; Beckman Coulter Immage analysator.三,Experimental methods(一) Samples: urine, weight, serum, liver, and tibiaUrine: Metabolic cage method.Seurm: By angular vein or abdomen cardinal vein.Liver: pathoscopy of organ.Tibia: pathoscopy of skeleton.(二)Observation of histomorphology1,Paraffin sectionFixation;Dehydration; Clearing;Dip to wax and embedding;Section.2,HE stainingDeparaffinage;Hydrqtion;Staining by haematoxylin;Differentiation by alclhol;Staining by eosin;Dehyudration;Clearing and mounting.四,Determination of CRP, Lc, C3, C4Assay method of CRP,C3 and C4: Immunoturbidimetry.Assay method of Lc: Flow cytometry.Principal of Beckman Coulter Immage: According to principal of fluid mechanics and apply the VCS techniques.五,Dental fluorosis graduationMonitor dental fluorosis every two weeks, which is recommended by geochemical disease laboratory.六,Statistics treatmentData were analyzed using SPSS 13.0 for Windows software. The results were expressed as the Mean±SD. Comparisons between groups were performed by repeated measure ANOVA, followed by Greenhouse-Geisser e correction factor, and followed by one-way ANOVA and LSD test and Tambane's T2 test, to detect significance of difference among multiple groups.α=0.05 indicated a statistically significant difference, andα=0.1 indicated size of test of homogeneity of variance.七,Quality control (一)Make sure the breeding and the environmental to normalize.(二)Controlling detection condition in order to warrant the equipments accurately and stablely during the experimental session.(三)Monitoring the changes of indexes during the detection process and deleting the abnormal data.(四)Blind method is used in the detection process.(五)Statistics methods should be used correctly.Results一,Dental fluorosis information60% of high-dose rats displays mild dental fluorosis at the end of the first month of experiment and 80% of high-dose rats displays medium dental fluorosis at the end of the second month of experiment and 6.7% of which displays severe dental fluorosis whereas 13.3% of low-dose rats displays medium dental fluorosis at the end of the second month of experiment. At the termination of experiment, all of the rats whose drinking water contains fluoride displays medium dental fluorosis and 20% high-dose rats displays severe dental fluorosis.二,Determinations of urine fluorineUrine fluorine increased with the dose of NaF, and there are significant differences among different dose-groups at the same period(P=0.000). While urine fluorine increased with the interval of experiment, and there are significant differences among different periods at the same dose-group(P=0.000).三,Determinations of serum fluorineSerum fluorine increased with the dose of NaF, and there are significant differences among different dose-groups at the same period(P=0.000). While serum fluorine increased with the interval of experiment, and there are significant differences among different periods at the same dose-group(P=0.000).四,Determinations of serum CRP Serum CRP increased with the dose of NaF, and there are significant differences among different dose-groups at the same period(F=8.019, P=0.009). It is appeared at the end of the first month of the experiment. While there are no significant differences among different periods at the same dose-group of serum CRP(F=0.939, P=0.346), and there are no interaction between the dosage and the interval(F=0.965, P=0.397).五,Determinations of serum C3Serum C3 decreased with the dose of NaF, and there are significant differences among different dose-groups at the same period(F=15.067, P=0.000). While there are significant differences among different periods at the same dose-group of serum C3(F=15.118, P=0.000), and there are interaction between the dosage and the interval(F=3.488, P=0.024).六,Determinations of serum C4Serum C4 decreased with the dose of NaF, and there are significant differences among different dose-groups at the same period(F=61.688. P=0.000). It is appeared at the end of the first month of the experiment. While there are significant differences among different periods at the same dose-group of serum C4(F=43.345, P=0.000), and there are interaction between the dosage and the interval(F=11.891, P=0.000).七,Determinations of serum LcSerum Lc increased with the dose of NaF, and there are significant differences among different dose-groups at the same period(F=45.472, P=0.000). It is appeared in the end of the first month of the experiment. While there are significant differences among different periods at the same dose-group of serum Lc(F=25.870, P=0.000), and there are interaction between the dosage and the interval(F=8.856, P=0.000).八,Findings with optical microscopeWith the dosage of NaF increasing, proximal tibia epiphyseal disk becomes more actively, and the bone trabecula becomes much thickness, even lead to constrictive type changes. While hepatocyte becomes swelling, degeneration and necrosis. Discussions一,Preparation for model of sub-chronic fluoride poisoning ratsDental fluorosis is the only symptom of chronic fluoride poisoning at earlier period, which can be observed at macroscopic level. It can reflect the degree of injury of the structure of tooth caused by fluorine especially and directly. Only at the end of the first month of the experiment, there are 60% of rats in high-dose group having the appearance of light-degree dental fluorosis. At the end of the second month, 80% of rats in high-dose group are in midrange-degree injure phase. Therefore, through the observation of dental fluorosis, the experiment can assess the duplication state of the model of sub-chronic fluoride poisoning.In the processes of many fluoride poisoning experiments of rats or rabbits, it can usually be seen that pathological changes develops fully in 2-3 months. After that period, as experiments go on, some pathological changes are not so obvious: on one hand, animals have accommodated to the high-fluorine circumstances; on the other hand, lots of earlier pathological changes get into recovery stage. So we use model of sub-chronic fluoride poisoning animals to carry out the experiment, and observe the levels of RP, C3, C4, Lc of the animals 1 time every 1 month, three months continuously. So that we can master the changing characteristics of the level of fluorine poisoning animals' sarcode inflammatory factor as the experiment goes on, to assess whether there is inflammatory reaction in the process of fluorine poisoning.二,Changes of fluorine level and inflammatory factor levelIn the experiment, rats have the symptoms of fluorine poisoning. The fluorine levels of serum and urine rise as dosage of fluorine rises, and the difference has statistical significance. But the fluorine level of serum kept at a comparatively low-grade constantly during three months of fluorine affected cycle, this result verifies the bulk deposition of bone fluorine and accelerating evacuation of urine fluorine which made the level of blood fluorine kept constant, by this way, important organs can refrain from incursions caused by excessive fluoride.Through the experiment, we can see that serum CRP content of rat raises as the content of fluorine affected raises, at the end of the first month of the experiment, the difference between groups has already had statistical significance, but at the same time dental fluorosis of low-dose group is not obvious; after this, as fluorine-affected time goes, level of CRP raises gradually, hinting the existence of inflammation state, and this phenomenon can be deemed as earlier period serologic marker of fluorine-caused hypertrophic arthritis. During the course of chronic inflammatory reaction, level of CRP can be constant for long period, and the result of this experiment also verifies that level of CRP does not change with prolongation of fluorine-infected time in the same fluorine-infected group.In this experiment, we also find that both addiment C3 and C4 decrease as time and dose increase. But at the end of the first month of the experiment, only the difference of C4 has statistical significance, and till the end of the second month the difference of C3 has statistical significance, testifying that C4 is more sensitive than C3, also hinting that at the end of the first month, there is immune suppression. Excessive fluorine may activate classical pathway and alternative pathway, consuming addiment, which made the levels of C3 and C4 in blood lower. In addition, the result of experiment shows that changes of C4 are more sensitive than dental fluorosis and display positive result earlier, so changes of C4 also can be regarded as earlier serology mark of fluorine poisoning injuries. And changes of levels of C3 and C4 also hints the existence of inflammatory reaction in the fluorine poisoning process.The proportion of Lc increases as fluorine-infected time and dose increases, at the end of the first year of the experiment the differences between groups have statistical significance, also hint the existence of chronic inflammatory reaction in organ.三,Pathological changesThrough this experiment, we observed that most of rats' hepatic cells of fluorine-infected group swelling, appearing disvolution, occasionally appearing necrosis hepatic cell, similar as the result of experiments before. And these pathological changes are fairly consistent with the pathological process of inflammatory reaction. Besides, under light microscope, we observed that fluorine-infected rats have cortical bone thickening and bone trabecula hyperplasy, which are similar as previously reported arthrodial cartilage calcification, necrosis and cartilage hyperplasy observed under X-ray, hinting that inflammation exists.ConclusionsWith fluoride dose increased and time prolonged, CRP and Lc levels were increased gradually, whereas C3 and C4 were decreased gradually, and C4 was more sensitive than C3. In the end of the first month of fluorosis, CRP,Lc and C4 changed in fluoride-treated group rats, especially in the low-dose group rats. This implied that CRP,Lc,C3 and C4 might be the serum marker of inflammation in fluorosis.