| Objectives:There were two aims having been focused on in this study. Firstly, the key responsible genes located in various pathways of apoptotic signaling and the signaling mechanisms associated with them were to be determined among rats with nasopharyngeal cancerous lesion induced by an integrated carcinogenesis of Qi deficiency toxin with contamination pathogenesis resulting in abnormally changed apoptotic activities in the process of initiation and development of such a kind of pathogenesis of in terms of TCM. Then, to be explored were the main pathways of apoptotic signaling and the related responsible genes and intervenable targets in the associated main links with apoptotic activities in nasopharyngeal epithelia undergoing carcinogenesis during the modeling course, induced by the therapeutic effects of herbal medicine preparation Qi-Boosting Toxin-Resolving Formulae (QBTRF).Methods:This study was carried out in two steps as following.1. Exploration on the characteristics of apoptotic signaling pathways in the developing process of carcinogenesis in nasopharyngeal epithelia induced by N,N'-dinitrosopiperazine used among rats undergone exhausted swimming ("contaminating toxin due to Qi deficiency" pathogenesis)Included in this part of study were 56 SD rats, randomly divided into 3 groups, i.e. modeling group I (MG1,24 rats), modeling group II (MG2,24 rats) and healthy controlling group (HCQ 8 rats). The rats in MG1 were sub-cutaneously injected in axillary fossa with N,N'-dinitrosopiperazine (DNP) and 12-O-tetradecanoylphorbol (TPA) simply, those in MG2 were also injected with DNP and TPA in the same way but in combination with the animals undergone exhausted swimming to mimic the carcinogenesis-inducing effect of Qi deficiency toxin with contamination pathogenesis on nasopharyngeal epithelia, and ones in HCG were also sub-cutaneously injected in the same area with the same volume of dimethyl sulfoxide (DMSO) diluted with normal saline, twice a week and 28 times in total for all the animals. All the animals were put into death for taking tissue samples from the nasopharynx in a regular numbers of rats and regular intervals of period during the whole experimental course lasted for 400 days. Then, the tissue samples were cut into two pieces respectively, with one part fixed in 4%neutral paraformaldehyde for pathohistological evaluation, TUNEL (TdT-mediated x-dUTP nick end labeling) method on apoptotic index and SABC immunohistochemical study on PCNA index and the expressive activities of genes in the apoptotic signaling pathways, as Bcl-2, Bax, Cyt-C and Caspase-3 in the signaling route of mitochondrion and NF-KBp65 and IκB in the signaling route of NF-κB p65, and the other part kept in nitrogen for RT-PCR assaying on BCL-2 and Bax mRNA.2. Investigation on the intervening effects of Qi-Boosting Toxin-Resolving Formulae on the apoptotic signaling pathways in nasopharyngeal epithelia undergone the developing process of carcinogenesis induced by N,N'-dinitrosopiperazine used among rats undergone exhausted swimming ("contaminating toxin due to Qi deficiency")Included in this part of study were 72 SD rats, randomly divided into 3 groups, i.e. modeling group (MQ 24 rats) with normal saline fed, experimental group (EQ 24 rats) with concentrated decoction of Qi-Boosting Toxin-Resolving Formulae (DQBTRF) fed and positive controlling group (PCQ 24 rats) with vitamin A acid fed, all treated for 90 days. All these animals were induced a carcinogenesis process in nasophayngeal epithelia with the procedures as the same as that in part A and intervened with the associated methods for a given period of time. Then, the animals were put into death in the same way as in part A to collect tissue sample from the nasopharynx and determine the changes of indicators in nasophayngeal epithelia, also in the same as above.Results:The results obtained from the study were as following, stated in two parts as that in methods.1. The characteristics of apoptotic signaling pathways in the developing process of carcinogenesis in nasopharyngeal epithelia induced by N,N'-dinitrosopiperazine used among rats undergone exhausted swimming ("contaminating toxin due to Qi deficiency" pathogenesis)More typical and severe pathohistological changes could be observed in nasopharyngeal epithelia with the progressing of carcinogenesis-inducing program with the use of DNP and TEA among the animals undergone exhausted swimming, the effects mimicking contaminating toxin due to Qi deficiency pathogenesis on the target organ, with nasopharyngeal carcinoma (NPC) developed in the nasopharynx at last. The occurring rate of induced epithelia lesion was much higher among the animals of MG2 than that in MG1 during the same experimental stage, while there were no significant differences in the expressive activities of apoptosis-associated genes determined here between these two groups in a same pathological stage.At the lesion developing stages of normal nasopharyngeal epithelia and simple hyperplasia, there were no significant differences in apoptotic index and PCNA index between MG1 and MG2 (P>0.05). With the development of induced carcinogenesis in nasopharyngeal epithelia, apoptotic index was significantly decreased in the tissue samples of nasopharynx with atypical hyperplasia and cancerous transformation. Although there was no significant difference in the apoptotic index found between the stage of atypical hyperplasia and that of cancerous transformation, significant differences could be determined when compared this indicator of these two stages with that of normal nasopharyngeal epithelia stage and simple hyperplasia stage respectively (P<0.05).The expressive activities of PCNA were both significantly increased in atypical hyperplasia and cancerous transformation nasopharyngeal epithelia with significant difference between the two stages of pathology (P<0.05). Furthermore, there were also significant differences between different stages of pathology, when compared with that of normal nasopharyngeal epithelia or simple hyperplasia nasopharyngeal epithelia respectively (P<0.05). Moreover, could found be a significant negative correlation present between apoptotic index and PCNA expressive activity in this progress of lesion developing (r=-0.794, P<0.05).There were no significant differences found in the expressive activities of Bcl-2, Cyt-C and Caspase-3 at the pathological stages of normal nasopharyngeal epithelia and simple hyperplasia (P>0.05). However, an increased expressive activity could be determined in Bcl-2 either in atypical hyperplasia or in cancerous transformation tissue samples with the development of induced lesion in nasopharyngeal epithelia, though with no significant differences found in these two stages of pathology themselves (P>0.05). In contrast, there were significant differences present when compared them with that of normal nasopharyngeal epithelia and simple hyperplasia (P< 0.05). The expressive activities of Cytochrome-C and Caspase-3 were decreased in nasopharyngeal epithelia with the pathological changes of atypical hyperplasia or cancerous transformation. Also, no significant differences could be found in these two stages of pathology themselves (P>0.05), while significant differences were found when compared them with that of normal nasopharyngeal epithelia and simple hyperplasia (P<0.05). The expressing activity of Bax showed a gradually increased tendency with the progressing of induced cancerous lesion in the pharynx, while there were no significant differences found among any stages of pathology when comparison was made (P>0.05). Meanwhile, various associations could be found among these determined indicators from correlating analysis carried out among them, with very significant negative correlation of Bcl-2 with Cytochrome-C and Caspase-3 (r=-0.668 and-0.871, P<0.05) respectively and significant positive correlation of Cytochrome-C with Caspase-3 (r= 0.698, P<0.05) while without significant correlation of Bax with Bcl-2, Cytochrome-C and Caspase-3 (r=-0.157,0.196,0.334; P= 0.593,0.501,0.243) respectively. Furthermore, there was also a parallel tendency of expressing activity in either Bax mRNA or Bcl-2 mRNA with Bax or Bcl-2 itself respectively in any stage of pathology. As seen in the mitochondrion route of apoptotic signaling pathway, similar changes could be observed in the NF-κB route of signaling pathway. In the lesion developing stages of normal nasopharyngeal epithelia and simple hyperplasia, there were no significant differences between them in the expressive activities of NF-κB p65 and IκBα(P >0.05), while there was increased expressing activities of NF-κB p65 in the tissue samples of atypical hyperplasia and cancerous transformation nasopharyngeal epithelia, with very significant differences these two stages of pathology (P<0.05). Moreover, very significant differences could also be determined in the expressing activities of this protein, when compared these two stages with either normal epithelia stage or simple hyperplasia one (P<0.05). On the other hand, the expressing activity of IκBαwas decreased in either atypical hyperplasia or cancerous transformation of the nasopharyngeal epithelia, even though without significant difference present between these two stages of pathology (P > 0.05). When compared these data with either that of normal nasopharyngeal epithelia stage or that of simple hyperplasia stage, very significant differences were found among them (P<0.05 respectively). Once more, a negative correlation could be determined between the activity of NF-κB p65 and that of IκBαin the cancerously transformed nasopharyngeal epithelia (r=-0.541, P<0.05).2. The intervening effects of Qi-Boosting Toxin-Resolving Formulae on the apoptotic signaling pathways in nasopharyngeal epithelia undergone the developing process of carcinogenesis induced by N,N'-dinitrosopiperazine used among rats undergone exhausted swimming ("contaminating toxin due to Qi deficiency" pathogenesis) The rate of cancerously transformed nasopharyngeal epithelia was 0% (0/5) in the rats of EG receiving concentrated DQBTRF feeding, significantly lower than that of MG (86.3%,5/6) receiving normal saline feeding (P<0.05). Though such a rate was also the same (0%,0/5) as that of EG, all the animals in this group were gradually dead during the experimental period from 250 to 310 d, suggesting a very significantly toxic effect of vitamin A acid on these experimental animals.In the simple hyperplasia stage of induced cancerous transforming lesion in the nasopharyngeal epithelia, no significant differences were found in the apoptotic index and PCNA activity among the 3 groups of rats (P>0.05). Till the pathological stage of atypical hyperplasia, apoptotic index was significantly elevated (P<0.05) and PCNA activity was significantly declined (P<0.05) among the rats of EG when compared with that of MGAlso in the simple hyperplasia stage of induced cancerous transforming lesion in the nasopharyngeal epithelia, no significant differences were determined in the expressing activities of Bcl-2, Bax, Cytochrome-C and Caspase-3 (P>0.05), when compared among them respectively. At the pathological stage of atypical hyperplasia, the expressing activity of Bcl-2 was significantly lower (P<0.05) and the activities of Cytochrome-C and Caspase-3 were significantly higher in the rats of EG than that of MG (P<0.05). Though the expressing activity of Bax was higher in the rats of EG than that of MG, no significant difference was determined between them (P>0.05). As the same as above in part one, there was also a parallel tendency of expressing activity in either Bax mRNA or Bcl-2 mRNA with Bax or Bcl-2 itself respectively in any stage of pathology. No significant changes in the expressing activities of NF-κB p65 and IκB a could be determined among these 3 groups of rats (P>0.05) in the simple hyperplasia stage of induced cancerous transforming lesion in the nasopharyngeal epithelia, while significantly lower expressing activity of NF-κB p65 (P<0.05) and higher expressing activity of IκB a (P<0.05) were assayed in the rats of EG than that of MG.Conclusions:Four aspects can be concluded based on this study as followings.1. Qi-deficient status of physique can be the genetically determined susceptible factor of physique to cancerous transforming development in the nasopharynx induced by environmental carcinogenic factors. Once one with such a physique status is contacted with this kind of carcinogenesis-inducing reagent for a certain period of time, the carcinogenesis-developing process may be induced in this way in nasopharyngeal epithelia. Then, the hypothesis of "contaminating toxin due to Qi deficiency" pathogenesis can be established reasonably to explain the carcinogenesis responsible for nasopharyngeal carcinoma developing in the nasopharynx.2. The developing process of induced cancerous transforming in nasopharyngeal epithelia of rats should be closely associated with the greatly decreased activity of apoptosis and significantly increased activity of cell proliferating in the target organ, with its pathological mechanisms possibly involved in the abnormally changed apoptotic signaling pathways of mitochondrion route and NF-κB route.3. Such a cancerous transformation developing process, induced by "contaminating toxin due to Qi deficiency" pathogenesis, can be effectively blocked by the use of herbal medicine preparation of Qi-Boosting Toxin-Resolving Formulae. This blocking effect on the induced lesion of cancerous transformation in nasopharyngeal epithelia may be underlay by the mechanisms of it to effectively inhibit the significantly elevated proliferating potentiality and actively induce apoptotic activity among them. Furthermore, the inducing effect of the formulae on apoptotic activity of nasopharyngeal epithelia with the induced lesion may be brought about by via intervening the routes of mitochondrion and NF-κB in the signaling pathway of apoptosis.4. Based on the results of this study, it has been suggested that the hypothesis of "contaminating toxin due to Qi deficiency" pathogenesis in terms of TCM responsible for the cancerous transformation of nasopharyngeal epithelia can be more effectively verified by the mimicking procedures of cancinogenesis in nasopharyngeal epithelia induced by N,N'-dinitrosopiperazine used among rats undergone exhausted swimming to prepare animal model with this kind of nasopharyngeal lesion. Furthermore, the experiment based on the intervening therapy with herbal medicine Qi-Boosting Toxin-Resolving Formulae has provided much powerful evidences for establishing a therapeutic principle to such a lesion by means of boosting Qi to resolve toxin, other than to confirm the reasonability of such a hypothesis to explain the pathogenesis of the lesion through the very effective therapeutic effects on it. |
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