The Study On TCM Syndromes Distribution And Cell Cycle Regulation Mechanism Of Laryngeal Precancerous Lesions

ObjectiveLaryngeal cancer is one of the most common diseases in the head and neck cancer remains a serious threat to human life and health. Laryngeal precancerous lesions is the central link of becoming laryngeal cancer. It not only can worsen development transformed into cancer, but also can be benign reversal of the normal cells. Early detection and intervention can prevent its development to malignancy. Therefore, the strengthening study of laryngeal precancerous lesions, to deepen the understanding of cancer mechanism, has become one of the forefront and key of head and neck cancer prevention and control work.The preliminary study suggested that cell cycle regulation was adjusted by two major factors of positive and negative types. The balance between them once became broken, it could lead to tumorigenesis. This had similarities with diseases caused by imbalance of human yin and yang in TCM. Abnormal cell cycle regulation was an important prerequisite for tumor development, and phlegm and blood stasis was an important factor leading to tumor development. Pre-clinical study of modern TCM and my tutor had found better therapeutic effects of laryngeal precancerous lesions with blood circulation and phlegm resolving method. But the TCM etiology and pathogenesis of Laryngeal precancerous lesions, and the mechanism of phlegm and blood stasis in the development of laryngeal precancerous lesions remained the lack of scientific support and validation currently.Basing on the literature research, This research will investigate the characteristics of the four diagnostic methods and distribution of the types of syndrome on laryngeal precancerous lesions in the clinical aspects. We will also study the cell cycle regulation mechanisms in process of laryngeal precancerous lesions and Phlegm and Blood Stasis of it In the experimental aspects. The results will provide ideas and direction for the medicine in prevention and treatment of precancerous lesions, and the theoretical support and experimental basis for blood circulation and phlegm resolving method of laryngeal precancerous lesions in clinic.Methods1. Literature researchSearching through ancient literature database such as Chinese Medical Code from Hunan Electronic Audio and Video Publishing House, and Chinese ancient literature database from the Guangdong Provincial Hospital, and meanwhile through modern literature database such as Chinese biology and medicine database (CBM),Chinese biomedical journals in the database (VIP Information),Chinese national knowledge infrastructure database (CNKI) and PubMed or Medline database of American national center for biotechnology information (NCBI).We summarized the relevant provisions of the classical and modern literature, and summed up the etiology and pathogenesis of laryngeal precancerous lesions in the ancient, dialectical analysis of the characteristics and treatment methods, modern epidemiology, classification, diagnosis, treatment methods and progress.2. Clinical researchThis study collected clinical cases of the syndrome information of laryngeal precancerous lesions through the use of evidence-based medicine, clinical epidemiology, and other modern research methodology combined with TCM. Through the establishment of an epidemiological questionnaire, the four diagnostic information and epidemiological data of214cases of laryngeal precancerous lesions were collected. With Epidata3.1packages to establish a database for data entry and verification, SPSS17.0statistical software for data analysis, the result preliminary draw the important indicators of syndrome diagnosis of laryngeal precancerous lesions, syndrome distribution and epidemiological features.3. Experimental researchIn this study, using flow cytometry, we will detect expressions of cell cycle factors (Cyclin Dl, Cyclin E, p21and p16protein) on specimens of phlegm and blood stasis type of laryngeal precancerous lesions, Yin deficiency type of the Laryngeal precancerous lesions, vocal cord polyps and early laryngeal cancer as the control groups. Through analysis of dynamic changes of the cell cycle factor in vocal cord polyps→laryngeal precancerous lesions→early laryngeal cancer, and different histological types and pathological classification of laryngeal precancerous lesions and laryngeal cancer, It will be made clear that cell cycle control mechanisms in the process of laryngeal cancer and phlegm and blood stasis type of laryngeal precancerous lesions.Results1. Clinical research(1) Pathogenic factors:In group of laryngeal precancerous lesions, long-term excessive use of voice pronunciation accounted for74.8%, long history of smoking accounted for43.9%, drinking habits accounted for21.5%, improper diet accounted for27.6%, staying up too late to sleep accounted for9.3%, getting cold accounted for5.1%. Among the three pathological types, there is no significant difference.(2) The gender composition:There are60male cases and92female cases of chronic hypertrophic laryngitis (atypical hyperplasia),22male cases and10female cases of laryngeal papilloma,20male cases and10female cases of laryngeal keratosis (vocal cord leukoplakia), The sex ratio of the three groups are as follows:0.65:1,2.2:1and2:1, and there are more males than females in the latter two groups, the gender composition between the groups has difference which has statistic significance (P <0.05).(3)The frequency of common signs and symptoms:The frequency of the symptoms which occurred greater than10%were:hoarseness (mild hoarseness, moderate hoarseness), foreign body sensation of pharynx, dry and burning throat, sore throat after over saying, and the pain did not move, itchy throat and cough, profuse sticky phlegm, preference of clearing the throat. The frequency of the signs which occurred greater than10%were poor vocal cord closure, vocal cord congestion and hypertrophy, smooth tumor surface, nodular,papillary or leukoplakia change of the tumor. The frequency of the tongue coating and pulse which present greater than10%were dark tongue or with Petechiae, greasy tongue coating, wiry pulse, white tongue coating, slippery pulse, red tongue, yellow tongue coating, thin tongue coating, fat tongue with teeth marks, thready pulse, deep pulse. (4) Syndrome cluster analysis:According to the results of computer cluster analysis, and combined with expert opinion, it should be clustered into four categories:Class I:hoarseness, red tongue, yellow tongue coating, greasy tongue coating, slippery pulse, nodular or papillary tumor, poor vocal cord closure. Class Ⅱ:clearing throat habits, sore throat after over saying, and the pain did not move, foreign body sensation of pharynx, fat tongue with teeth marks, dark tongue or with petechia, deep pulse, vocal cords congestion and hypertrophy, smooth tumor surface. Class Ⅲ:mild hoarseness, profuse sticky phlegm, cough, itchy throat, white tongue coating, wiry pulse, normal vocal cord closure. Class IV: severe hoarseness, dry and burning throat, thin tongue coating, thready pulse, and tumor leukoplakia-like change.(5) The important index of syndrome diagnosis according to main symptom, secondary symptom, tongue coating and pulse, local signs in turn are as follows:①Syndrome of damp-heat stagnation main symptoms:hoarseness; tongue coating and pulse:red tongue, greasy tongue coating, yellow tongue coating, slippery pulse; local signs:vocal cords congestion and hypertrophy, poor vocal cord closure, nodular or papillary tumor.②Syndrome of qi deficiency and phlegm stasis main symptoms: hoarseness; secondary symptoms:preference of clearing voice, foreign body sensation of pharynx, sore throat after over saying, and the pain did not move; tongue coating and pulse:dark tongue or with petechia, greasy tongue coating, white tongue coating, wiry pulse, slippery pulse, deep pulse; local signs:vocal cords congestion and hypertrophy, smooth tumor surface.③Syndrome of turbid phlegm accumulation main symptoms: hoarseness(mild); secondary symptoms:profuse sticky phlegm, itchy throat and cough; tongue coating and pulse:white tongue coating, wiry pulse; local signs:congestive hypertrophy of the vocal cords, smooth tumor surface, normal vocal cords closure④Syndrome of hyperactivity of fire due to yin deficiency main symptoms:hoarseness(severe):dry and burning throat, foreign body sensation of pharynx; tongue coating and pulse:red tongue, white tongue coating, thin tongue coating, thready pulse; local signs:congestive hypertrophy of the vocal cords, nodular papillary or leukoplakia change of the tumor.(6) Syndrome distribution:Class Ⅰ:syndrome of damp-heat stagnation; Class Ⅱ:syndrome of qi deficiency and phlegm stasis; Class Ⅲ:syndrome of turbid phlegm accumulation; Class Ⅳ:syndrome of hyperactivity of fire due to yin deficiency. among which syndrome of damp-heat stagnation are49cases, accounting for22.9%; syndrome of qi deficiency and phlegm stasis are87cases, accounting for40.7%; syndrome of turbid phlegm accumulation are23cases,10.7%; syndrome of hyperactivity of fire due to yin deficiency are55cases, accounting for25.7%.(7) The syndrome distribution factors as follows:①The relationship between the Traditional Chinese Medicine syndrome and gender:Male accounted for a large percentage (67.3%) for the syndrome of damp-heat stagnation, while the female accounted for a large percentage (respectively58.6%and65.2%,54.5%) for the syndrome of qi deficiency and phlegm stasis, turbid phlegm accumulation, hyperactivity of fire due to yin deficiency of which has statistic significant differences (P<0.05).②The relationship between the Traditional Chinese Medicine syndrome and age:Qi deficiency and phlegm and blood stasis account for a large percentage (42.9%) of the laryngeal precancerous lesions of the middle-aged patient,other syndromes are relatively dispersed; each type in the youth group and older group were relatively scattered, it has no statistic significance (P>0.05).③The relationship between the Traditional Chinese Medicine syndrome and duration:Difference between the average onset time of syndrome types of laryngeal precancerous lesions (28.17to37.67) was small, and it has no statistic significance (P>0.05)④The relationship between the Traditional Chinese Medicine syndrome and pathological types:Qi deficiency and phlegm and blood stasis type was common in chronic hypertrophic laryngitis and laryngeal papilloma (accounting for40.1%,56.3%). Hyperactivity of fire due to yin deficiency type was common in laryngeal keratosis(accounting for33.3%). But it has no statistic significance (P>0.05)⑤The relationship between the Traditional Chinese Medicine syndrome and pathogenic factors:History of smoking and drinking were common in damp-heat stagnation and Qi deficiency and phlegm and blood stasis types, and i t had statistic significance (P<0.05). Improper diet and staying up too late to sleep were common in Qi deficiency and phlegm and blood stasis types, and long-term excessive use of voice and getting cold were common in Qi deficiency and phlegm and hyperactivity of fire due to yin deficiency types, but those had no statistic significance (P>0.05)⑥The relationship between the Traditional Chinese Medicine syndrome and local signs:Smooth tumor surface was related to Qi deficiency and phlegm and blood stasis type (44.9%), nodular or papillary tumorn related to damp-heat stagnation (47.1%), leukoplakia change of the tumor related to hyperactivity of fire due to yin deficiency types (68.8%).They had statistic significance (P<0.05)⑦The relationship between the Traditional Chinese Medicine syndrome and degree of hoarseness:The hoarseness degree of voice came from the score of disability index in VHI-10questionnaire. There were50cases of mildly abnormal patients,150cases of moderately abnormal cases, six of the normal cases, eight of severe disorders cases, which had not statistically significant (P>0.05).(8) Rate of recurrence and malignant transformation:In this study,137cases of laryngeal precancerous lesions were followed up. There were15cases of recurrence, four cases of vocal cord leukoplakia, four cases of laryngeal papilloma,7cases of vocal cord polyps with mild to moderate dysplasia. Recurrence rate of laryngeal precancerous lesions was10.95%. Among those, six cases of mal ignant transformation, four cases of vocal cord leukoplakia, one cases of laryngeal papilloma, and one case of squamous moderate atypical hyperplasia in vocal cord. Malignancy cases have years of history of smoking and drinking history. Malignant transformation rate of laryngeal precancerous lesions was4.38%.2. Experimental research(1) Expressions of Cyclin D1had a progressive enhancement from vocal cord polyps, laryngeal precancerous lesions to Laryngeal cancer(the percentage as follows:13.04%,20.29%,42.42%). However, all of the fluorescence intensity are weak. There were statistical differences in pairwise comparisons (P<0.05). The result of mild dysplasia, moderate dysplasia and severe dysplasia in laryngeal precancerous lesions had an up trend of positive expressions, which were13.95%,28.57%,31.58%, but there was no significant difference (P>0.05). Expressions of Cyclin D1 in epithelium atypical hyperplasia of different types(chronic hypertrophic laryngitis), laryngeal papilloma and laryngeal keratosis were19.51%,21.05%,22.22%, respectively. There was no statistical significance among the three (P>0.05). The positive expression rates of Cyclin D1in well differentiated was40.00%, moderately differentiated41.67%and poorly differentiated43.75%in different pathologic types of laryngeal carcinoma, and carcinoma in situ was35.71%, invasive carcinoma47.37%in different pathological grade of laryngeal carcinoma. Both showed an increasing tendency, without statistical significances (P>0.05)(2)Expressions of Cyclin E had a Progressive enhancement from vocal cord polyps, Laryngeal precancerous lesions to Laryngeal cancer(the percentage as follows:26.09%,43.48%,93.94%). All of the fluorescence intensity are strong. There were statistical differences in pairwise comparisons (P<0.001). The result of mild dysplasia, moderate dysplasia and severe dysplasia in laryngeal precancerous lesions had an up trend of positive expressions, which were36.59%,52.63%,55.56%, but there was also no significant difference (P>0.05). Expressions of Cyclin D1in epithelium atypical hyperplasia of different types(chronic hypertrophic laryngitis), laryngeal papilloma and laryngeal keratosis were25.58%,57.14%and78.95%, respectively. It showed an increasing tendency, and there was statistical significance among the three (P<0.001).The positive expression rates of Cyclin Dl in different pathologic types and grade of laryngeal carcinoma were high (80%～100%), a rising trend. But there were no statistical significance (P>0.05)(3) Expressions of P16had a diminishing gradient phenomenon from vocal cord polyps, laryngeal precancerous lesions to Laryngeal cancer (the percentage as follows:60.87%,40.58%,15.15%). There were statistical differences in pairwise comparisons (P<0.05). The result of mild dysplasia, moderate dysplasia and severe dysplasia in Laryngeal precancerous lesions had an downward trend of positive expressions, which were51.16%,42.86%,15.78%, and had significant difference (P<0.05). Expressions of P16in epithelium atypical hyperplasia of chronic hypertrophic laryngitis, laryngeal papilloma and laryngeal keratosis were41.46%,42.11%,33.33%, respectively. There was no statistical significance (P>0.05). The positive expression rates of P16in high, mediate and low differentiated laryngeal carcinoma were20.00%,25.00%,12.50%, and in different pathological grade of laryngeal carcinoma, carcinoma in situ35.71%and invasive carcinoma47.37%.Both showed no statistical significances (P>0.05)(4) Expressions of P21had a diminishing gradient phenomenon from vocal cord polyps, laryngeal precancerous lesions to Laryngeal cancer (the percentage as follows:47.83%,26.09%,24.24%). But there were no statistical differences in pairwise comparisons (P>0.05).The result of mild dysplasia, moderate dysplasia and severe dysplasia in Laryngeal precancerous lesions had an downward trend of positive expressions, which were27.91%,28.57%,21.05%.Expressions of P21in epithelium atypical hyperplasia of chronic hypertrophic laryngitis, laryngeal papilloma and laryngeal keratosis were26.83%,26.32%,22.22%, respectively. Both had no significant difference (P<0.05). The positive expression rates of P21in high, mediate and low differentiated laryngeal carcinoma were20.00%,25.00%,18.75%, and in different pathological grade of laryngeal carcinoma, carcinoma in situ21.43%and invasive carcinoma21.05%.Both showed no statistical significances (P>0.05)(5) The positive rates of CyclinDl and CyclinE expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively high(23.40%and46.81%respectively), while in Yin Deficiency type relatively low(9.09%and36.36%respectively). There was no statistical significance (P>0.05). The positive rates of P16expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively low (34.04%), while in the Yin deficiency type relatively high(45.0%). Still, no statistical significances showed (P>0.05). However, the positive rate of expression of P21in the phlegm and blood stasis type and Yin Deficiency type was opposite (27.66%and23.0%respectively), and had no statistical significances (P>0.05)Conclusion1. The epidemiological characteristics of laryngeal precancerous lesions(1)The risk factors of laryngeal precancerous lesions were followed by improper using of voice, smoking, drinking, improper diet, staying up late, colds.(2) The sex ratio of man and women in laryngeal papilloma and laryngeal keratosis were2.2:1and2:1, while0.65:1in chronic hypertrophic laryngitis.(3) Recurrence rate of laryngeal precancerous lesions was10.95%, and the malignant transformation rate was4.38%.2. TCM pathogenesis, syndrome distribution and epidemiological features(1)TCM pathogenesis as follows:The outside pathogenesis was damp-heat stagnation and toxic evil due to smoking, drinking and Exopathy. The Internal ones were phlegm stagnation from spleen injury due to improper diet, and deficiency of vital qi and yin due to staying up too late and long-term excessive use of voice.(2) In syndrome distribution according to the rate from large to small, there were four types as follows:damp-heat stagnation syndrome, qi deficiency and phlegm stasis syndrome, turbid phlegm accumulation syndrome, hyperactivity of fire due to yin deficiency syndrome. The important index of syndrome diagnosis can be found in the part of results (abbreviated).(3)Factors related to the syndrome distribution as follows:male common in the syndrome of damp-heat stagnation type, while the female common in the syndrome of qi deficiency and phlegm stasis, turbid phlegm accumulation, hyperactivity of fire due to yin deficiency types;history of smoking and drinking common in damp-heat stagnation and Qi deficiency and phlegm and blood stasis types. There were significant differences in the local signs of TCM syndromes (abbreviated). Pathological type, age, duration of disease, hoarseness extent had no significant effect for TCM syndrome distribution of laryngeal precancerous lesions.3. mechanisms of cell cycle control(1) The expressions of CyclinDl and CyclinE in vocal cord polyps→laryngeal precancerous lesions→laryngeal cancer, and in mild dysplasia→moderate dysplasia→severe dysplasia groups of Laryngeal precancerous lesions showed a gradual increase of the gradient phenomenon. Statistically, there were significant differences, especially the first group.It prompted that the over-expression of the positive regulatory factor of CyclinDl and CyclinE leaded cell cycle to accelerate the malignant proliferation. Expressions of CyclinDl and CyclinE in different types and pathologic grading of laryngeal cancer, did not show obvious differences, which may be related to the less specimen. Generally, expression of CyclinE in the highly malignant lesions was higher than CyclinD1.CyclinE would play a greater role in promoting the restriction point of G1/S than CyclinD1, which still needs further study.(2) The expressions of P16and P21in vocal cord polyps→laryngeal precancerous lesions→laryngeal cancer, and in mild dysplasia→moderate dysplasia→severe dysplasia groups of Laryngeal precancerous lesions showed a diminishing gradient phenomenon. But only the group of P16showed significant difference statistically. It prompted that the tumor suppressor gene of P16played an important role in the inhibition of Rb phosphorylation mediated by CDK4/CDK6to prevent the process of abnormal cells in G1/S. In different types and tumor grades of laryngeal cancer, positive rates of P16and p21expression was lower. But no significant difference was between the two groups, still related to the limited sample size.(3)The positive rates of CyclinD1and CyclinE expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively high, while in Yin Deficiency type relatively low. The positive rates of P16expression in phlegm and blood stasis type of laryngeal precancerous lesions were relatively low, while in the Yin deficiency type relatively high. Comparison of P16, the positive rate of expression of P21in the phlegm and blood stasis type and Yin Deficiency type was opposite. But the above expressions between the two groups showed no significant difference.It prompted that in the process of disease caused by phlegm and blood stasis, high expression of positive regulatory factor of CyclinD1and CyclinE, and low expression of negative regulatory factor P16leaded to abnormal cell cycle regulation might be the molecular biology mechanism. However, it needs to be verified by further in-depth study of expanding the sample size.Deficiencies and ProspectsThe study still had many deficiencies, such as limitted research information including insufficient sample size and non-multi-center study, and the insufficient study design in both clinical research table content and the selection of specimen collection method in experimental study. There was no any clear basis for Chinese Medicine of the cellular and molecular biological mechanism from the research results by now. The research study should be gradually modified and improved in the future clinical and experimental practice.