Study On The Relationship Between Constitution And Cervical Tissue Protein Expression In Patients With HR-HPV Infection And The Mechanism Of Action Of Heat-clearing And Dehumidifying Ge

Objective1.To explore the distribution law of the main traditional Chinese medicine(TCM)constitution types of the patients with HR-HPV infection cervical lesions,and analyze the relationship between the clinical characteristics and constitution types,in order to provide a theoretical basis for the prevention and treatment of HR-HPV infection and cervical lesions.2.The expression of apoptotic proteins(Bcl-2,Bax,Caspase-3)at each stage of cervical lesions and their relationship with the main constitution types were analyzed to preliminarily explore the main constitution types of HR-HPV infected patients with cervical lesions,providing ideas and references for the prevention and treatment of HR-HPV infection and cervical lesions with TCM to prevent the occurrence of CC.3.Based on clinical research,animal models of tumor bearing mice were constructed by using HR-HPV16 positive SiHa cells.The antitumor efficacy of "Qingre dampness gel"were studied,and the mechanism of tumor suppression was preliminarily discussed.Method1.Constitutional research:The general data and main clinical symptoms of 230 patients with cervical diseases(including 49 cervicitis patients,72 LSIL patients,100 HSIL patients and 9 CC patients)infected by HR-HPV were collected by questionnaire survey.The general characteristics and high-risk factors of patients with cervical diseases infected by HR-HPV were explored,and the distribution law of cervical disease level and physique type was analyzed.2.Relationship between apoptosis-related proteins and TCM constitution:Based on the above results,74 patients with cervical diseases were selected,including 21 patients with cervicitis(8 with Damp-heat constitution’s with Yang deficiency constitution and 5 with Qi deficiency constitution),24 patients in LSIL group and 24 patients in HSIL group(8 with Damp-heat constitution,8 with Yang deficiency constitution and 8 with Qi deficiency constitution),5 patients with CC(3 with Damp-heat constitution,1 with Yang deficiency constitution and 1 with Qi deficiency constitution).The expression of Bcl-2,Bax and Caspase-3 in cervical lesions was detected by immunohistochemistry,and the relationship between the expression of Bcl-2,Bax and Caspase-3 and the degree of cervical lesions and the TCM constitution was analyzed.3.Experimental research:Establish animal model of HR-HPV 16 positive SiHa cell bearing tumor,observe and study the antitumor effect of"Qingre dampness gel"alone and combined with cisplatin on tumor,and analyze the tumor tissue,lung,liver,spleen and kidney by HE staining,Immunohistochemistry,TUNEL and Western blot were used to detect the apoptosis and the expression of Bcl-2,Bax,Caspase-9 and Caspase-3 in tumor tissues,and to analyze the possible mechanism of action of the drugs.Results1.Constitutional research:The constitution types of 230 patients with cervical lesions infected by HR-HPV were mainly biased constitution,and the proportion was from high to low:Damp-heat constitution(26.09%),Yang deficiency constitution(21.30%),Qi deficiency constitution(17.82%),Normal constitution(13.49%),Qi-stagnation constitution(10.43%),Yin deficiency constitution(5.65%),Phlegm-dampness constitution(3.04%),Blood-stasis constitution(1.74%)and special constitution(0.43%).Different levels of cervical lesions,different ages and different types of HR-HPV infection have differen constitution distribution and proportion.2.Relationship between apoptosis-related proteins and TCM constitution:(1)The relationship between apoptosis-related proteins and cervical lesion level:Bcl-2 protein expression was increased with the deepening of cervical lesions,while the expression of Bax and caspase-3 decreased with the deepening of cervical lesions.(2)The relationship between apoptosis-related proteins and TCM constitution:The expression of Bcl-2,Bax and caspase-3 proteins was different among patients with different constitution types of cervical lesions,the expression of Bcl-2 protein was the highest in the patients with Damp-heat constitution,followed by those with patients with Yang deficiency constitution and Qi deficiency constitution.Bax and Caspase-3 protein expression levels were the lowest in patients with Damp-heat constitution,followed by those with patients with Yang deficiency constitution and Qi deficiency constitution.(3)The expression of Bcl-2 protein was enhanced with the increase of the degree of lesions in patients with Damp-heat constitution,while the expression of Bax and Caspase-3 protein was decreased with the increase of the degree of lesions.3.Experimental research:(1)After the intervention,tumor growth was inhibited in the"Qingre dampness gell"group,cisplatin group,combination therapy group compared with model group and carbomer 940 group,the inhibition was most obvious in the combination group.(2)The "Qingre dampness gel"group did not cause damage to liver and kidney function of tumor bearing mice,the liver and kidney function of tumor bearing mice were damaged to different degrees in both cisplatin group and combination therapy group,the injury was more obvious in the cisplatin group.(3)HE staining showed that the tumor necrosis in the "Qingre dampness gell" group,cisplatin group and combination therapy group was obvious,the most obvious necrosis was combination therapy group,no tumor metastasis was found in lung,liver,spleen,kidney and other organs.(4)Immunohistochemistry,TUNEL and Western Blot were used to analyze the results,"Qingre dampness gel",cisplatin group and combination therapy group can reduce the expression of Bcl-2 protein."Qingre dampness gel",cisplatin group and combination group could inhibit the growth of tumor by downregulating the expression of Bcl-2 protein and up regulating the expression of Bax and Caspase-3 protein.The combination therapy group had a more obvious change,and its mechanism might be achieved by regulating mitochondrial apoptosis pathway.Conclusions1.Constitutional research:Patients with cervical lesions caused by HR-HPV infection mainly had a biased constitution,especially in Damp-heat constitution,Yang deficiency constitution,Qi deficiency constitution.The distribution of constitution is related to the grade of cervical lesions,age,frequency of HR-HPV infection.2.Relationship between apoptosis-related proteins and TCM constitution:The local expressions of Bcl-2,Bax and Caspase-3 in patients with cervical lesions caused by HR-HPV infection were different in different cervical lesion levels and different constitution types.The Damp-heat constitution may be a more relevant type of constitution in patients with cervical lesions infected with HR-HPV compared with Yang deficiency constitution,Qi deficiency constitution.3.Experimental research:"Qingre dampness gel"can inhibit the growth of tumor bearing mice,and has no obvious damage to liver and kidney function.The expression of Bcl-2,Bax and Caspase-3 proteins related to mitochondrial apoptotic pathway may be the molecular mechanism of its anti-tumor effect.