Effects Of Kang-Jia-Wan On Angiogenesis And Thyrocyte Apoptosis Induction And Proliferation Inhibition In Rats Of Goiter

BackgroundGoiter,defined as benign thyroid enlargement,is a common disease that occurs in more than ten percent population all over the world,which has different subtypes,for instance,simple goiter,nudolar goiter and toxic goiter.Its etiololy is complex. Environmental factors play an important role in the formation of goiter,such as iodine deficiency,iodine excess,smoking and drugs.Besides,a few gene dysfunctions contributed to the development of goiter have been discovered in recent years for example,thyroglobulin,sodium/iodide symporter,thyroid peroxidase and thyrotropin receptor.In addition,goiter genesis is related to immune,infections and gender.Angiogenesis is involved in the formation and development of goiter.Angiogenesis is the sprouting of new blood vessels from pre-existing capillaries.It is a complex process tightly controlled by a varity of angiogenesis stimulators,of which VEGF,FGF and IGF-1 play an important role.In thyroid the activation of TSH receptor is able to up-regulate the expression of VEGF and IGF-1,which cause angiogenesis.Similarly, FGF,a central angiogenesis stimulator,is also a factor involved in goiter.Premilitary clinical study showed that angiogenesis was closely correlated with toxic goiter,in which the microvessel count and the expression of VEGF as well as IGF-1 were extensively increased.Goiter results from the imbalance between proliferation and apoptosis of thyrocytes leading to insufficient apoptosis,which is crucial for goiter formation.Apoptosis,i.e. programmed cell death,is an exactly regulated process controlled mainly through two pathways.The extrinsic pathway is activated by the interaction of death ligand(DR) with its cell surface death receptors including Fas,TNFαand TNF-related apoptosis-inducing ligand(TRAIL).Through them the extracellular signals are transduced into intracellular receptor leading to start apoptosis.The intrinsic pathway can be activated by a variety of stimuli which lead to the permeabilization of the mitochondrial outer membrane and release of cytochrome C,which activates apoptosis. A number of studies demonstrated that apoptisis abnormalities were involved in goiter. It turned out that Fas is expressed on the cell memberane of thyrocytes.And Fas expression was significantly increased in Graves'disease and nontoxic multinodolar goiter.Essentially,goiter is of thyrocytes over-proliferation in histology.The pathway of Thyroid Stimulating Hormone(TSH) and its receptor plays a key role in stimulating thyrocyte proliferation.Therefore,a deficiency in either thyroid hormone synthesis or intake leads to an increased TSH production by a feedback way,which causes thyrocyte proliferation and goiter.Graves' disease is an autoimmune disorder,in which the body produces antibodies to the receptor for TSH.These antibodies can cause thyrocyte over-hyperplasia through mimicing TSH effects.It has been thought that the sensibisity enhancement of thyrocyte to TSH is responsible for the genesis of simple goiter and nodular goiter,in which the thyroid hormones do not alter apparently.But in recent years,the role of a rarity of growth factors promoting cell proliferation in goiter was revealed,such as IGF-1 and HGF.To date,the treatment of goiter is mainly composed of three methods:thyroxin administration,surgery and ¹³¹I radiotherapy.The three therapies have their complications respectively,e.g.return to pretherapy values in measurement of thyroid size,bone loss,hypothyroidism and cardiac changes.Therefore,better therapy need to be explored.Traditional Chinese Medicine has more than two thousand years history which plays an important role in China.Kang-Jia-Wan(KJW) is a patented(patent No. CN1271593) and approved hospital preparation of Traditional Chinese Medicine that has been used for years for the treatment of a variety of goiter.However,the underlying mechanism remains unclear.Thereforce,the mechanism of KJW treating goiter was investigated in three parts:antiangiogenesis,inducing apoptosis and inhibiting cell proliferation.Objectives1 To observe the effects of KJW on angiogenesis using CAM model.2 To explore the effects of KJW on thyrocyte apoptosis in goiter of rats given MMI.3 To explore the inhibitory effects of KJW on thyrocyte proliferation in goiter of rats given MMI.Methods1 CAM angiogenesis modelAfter 3-d incubation at 37℃,fertilized white Leghorn eggs were cracked,and chick embryos with intact yolks were carefully placed in 10×10cm Falcon dishes.After 2 days of incubation in 4%CO2 at 37℃,filter paper disks containing Kang-Jia-Wan were implanted on the CAM of individual embryos.After 5 days of incubation,CAMs were examined for the number of vascular nodal points around the field of the implanted disks by using a stereoscope to take photographs.2 Goiter animal model and groupingMale Wistar rats(weighing 180-220 g) were separated into four groups at random: normal,MMI,LKJW and HKJW.The normal group received normal rat chow and plain tap water and the other three groups were given MMI in drinking water.One week later,the LKJW and HKJW groups were orally administrated with KJW at 250mg/kg or 1000mg/kg(equal to four times,twenty times clinical dosages) once a day for 12 weeks.At the end of the 12 weeks' treating with KJW,the rats were sacrificed under pentobarbital anaesthesia.The thyroid glands were devided into three parts:one was fixed with 4%paraformaldehyde;another was fixed with 3%glutaraldehyde for electron microscopy;the third was freezed in liquid nitrogen for western blotting.3 Histological methodsThe methods including HE staining,electron microscopy,TUNEL staining and immunohistochemistry were used.The results were analyzed and quantified with the softwire Image-Pro Plus 6.0.4 Western Blotting Results1 No derect inhibitory effects of KJW on angiogenesis in CAM model were observed.2 MMI successively induced goiter of rats whose serum levels of TT3 and TT4 were significantly lower than that in normal group.The level of serum TSH in MMI,LKJW and HKJW groups was greatly higher than in normal group.Moreover,KJW did not significantly influence thyroid function.3 The thyroid weight relative to body weight(mg/100g body weight) was significantly higher in the MMI group compared to the normal group.Compared with the MMI group,the relative thyroid weight was decreased by 10%in the LKJW group (p=0.067) and by 21%in the HKJW group(p＜0.05).No significant difference was found between the LKJW and HKJW groups(p=0.052).4 At least 90%thyroid follicles in MMI group were lost and the colloid substance was almost absent compared to the normal group.Furthermore,the cell density per mm² increased in MMI group compared to the normal group.By comparison,with 12 weeks treatment of high-dose KJW,the follicular area and cell density restored partly significantly compared to the MMI group.On electron micrographs,representative signs of apoptosis were observed in the KJW treated groups,such as nuclear karyorrhexis,condensed chromatin and vacuolation of the cytoplasm of thyrocytes.5 The TUNEL assay showed that the number of positively stained thyrocytes was higher in the two KJW treatment groups compared to the normal and MMI groups. Further quantitative analysis indicated significant increases of TUNEL positive cells in KJW treated groups compared to MMI group(3.2±1.3,6.5±2.7 vs.0.58±0.78 respectively,p＜0.01).In addition,quantitative analysis showed that the number of apoptotic cells in the HKJW group was one-fold higher than that in the LKJW group(p＜0.05).6 Quantitative morphological analysis of the immunohistochemistry for activated caspase-3 showed that both the low- and high-dose KJW resulted in an increase in the levels of caspase-3 activity relative to either the normal or MMI alone treatment group (p＜0.05,respectively).In addition,KJW treatment at high dose markedly increased the positive cells compared to low-dose KJW treatment(p＜0.05).The results of the activated caspase-3 displayed the same changes with the immunohistochemistry results. Additionally,the expression of caspase-3 protein(35KD) was detected in the samples of the four groups.But,a strong band was seen in the samples derived from the rats treated with KJW relative to these rats without KJW treatment.Compared with the normal group,the caspase-3 expression was increased to 3.7-fold,4.6-fold and 6.3-fold in MMI,LKJW and HKJW groups,respectively.7 Western blotting analysis of Fas showed that,compared with the MMI group,the Fas protein expression was increased by 45%(p＜0.05) and by 103%(p＜0.05) in the LKJW and HKJW groups.8 KJW decreases the PCNA expression in goiter at protein levelThe PCNA immunohistochemistry quantitative analysis indicated significant decreases of PCNA positive cells in KJW treated groups compared to MMI group (10.83±3.87,6.49±2.61 vs.16.56±4.25 respectively,p＜0.05).In addition, quantitative analysis showed that the number of PCNA positive cells in the H KJW group was markedly lower compared with the LKJW group(p＜0.05).Western blotting results for PCNA were consistent with those data obtained from immunohistochemistry staining of PCNA.The expression level of PCNA in the MMI group was strongly increased compared with the normal group(p＜0.05).But compared with the MMI group,the PCNA protein expression was decreased by 45%(p＜0.05) and by 58%(p＜0.05) in the LKJW and HKJW groups.These data showed that KJW decreased the PCNA expression on a dose-response-like fashion.9 KJW decreases the cyclin D1 expression in goiter at protein levelThe results from western blotting showed that the bands representing the expression of cyclin D1 were detected in the samples of the four groups.A strong band was seen in the samples derived from the rats in the MMI group relative to these rats in the normal group.Compared with the MMI group,the cyclin D1 expression was decreased by 25% and 39%in the LKJW and HKJW groups,respectively.These data showed that KJW decreased the cyclin D1 expression in a dose-dependent manner.Conclusions 1 KJW does not have the function of antiangiogenesis in CAM.2 The treatment of KJW decreases thyroid index in rat goiter induced by MMI.3 The treatment of KJW improves thyroid morphology in rat goiter induced by MMI.4 The treatment of KJW induces apoptosis of thyrocytes and upregulates the expression of activated caspase-3 and Fas in rat goiter induced by MMI.5 The treatment of KJW inhibits thyrocytes proliferation on a dose-response fashion in rat goiter induced by MMI.