Mechanism Research On The Effect Of Kidney-Tonifying Method In Inhibiting Female Lung Adenocarcinoma Based On MiR-1-3p

Lung adenocarcinoma(LAD)is the most common type of lung cancer,with a higher incidence in non-smokers.Studies have shown that the incidence of LAD in men is stable,but it is still increasing in women.Due to gender differences,sex hormones are believed to play an important role in the occurrence of lung cancer.Many previous studies have also shown that female lung adenocarcinoma is related to sex hormone levels.The combined research of modern medicine and traditional Chinese medicine shows that the "hypothalamus-pituitary-gonadal axis" can be regarded as one of the essences of the kidney.Patients with kidney deficiency usually have different degrees of dysfunction of the hypothalamic-pituitary-gonadal axis.Sex hormone levels are closely related.According to the important role of sex hormones in the occurrence and development of female lung cancer,the relationship between the two is understood from the perspective of traditional Chinese medicine,and it is believed that there may be a certain correlation between kidney deficiency and female lung adenocarcinoma.With its unique overall concept,Chinese medicine therapy has obvious advantages in improving patient symptoms,enhancing curative effect,reducing toxic and side effects,improving quality of life and prolonging patient survival.In recent years,it has been widely used in cancer treatment.Combined with the clinical characteristics of patients with lung adenocarcinoma,many physicians have achieved good clinical results using methods such as invigorating the kidney and cultivating energy,nourishing temperature and removing dampness.At present,many studies have expounded the mechanism of Yanghe decoction in the treatment of breast cancer,but there is still a lack of extensive and in-depth research in lung adenocarcinoma,and further research is needed to provide a theoretical basis for its clinical application.Therefore,this topic is devoted to studying the distribution law and characteristics of TCM syndrome elements in female lung adenocarcinoma.Under the guidance of the method of tonifying kidney and warming yang,Yanghe decoction is selected as the basic formula,and its inhibitory effect on lung adenocarcinoma and its related mechanism are discussed through experimental research.The research content is mainly divided into the following two parts:1.Clinical researchThrough statistical analysis of the information on the four diagnosis of traditional Chinese medicine in female lung adenocarcinoma patients,the syndrome manifestations of female lung adenocarcinoma patients were summarized,and the distribution law and characteristics of TCM syndrome elements were discussed,and their correlation with age,smoking history,family history,menopause age,hormones level,clinical stage and other factors.2.Experimental researchThe inhibitory effect of Yanghe Decoction on lung adenocarcinoma cells was observed by in vitro experiments;An animal model of lung adenocarcinoma transplanted with kidney deficiency syndrome in female nude mice was constructed,and the inhibitory effect of YangheDecoction on transplanted tumors was observed by in vivo experiments;Combined with network pharmacology to predict its molecular mechanism of action,and conduct experiments to verify its regulatory mechanism.Part Ⅰ Clinical study on the distribution of TCM syndrome elements in female lung adenocarcinoma patientsObject1.To explore the distribution law and characteristics of TCM syndrome elements in female lung adenocarcinoma patients;2.Statistical analysis of the relationship between age,smoking history,family history,age at menopause,hormone level,clinical stage and syndrome elements in female lung adenocarcinoma patients.Content and MethodThe data of female patients diagnosed with lung adenocarcinoma in Qingdao Haici Medical Group and Shandong Provincial Hospital of Traditional Chinese Medicine from July 2020 to December 2021 were collected,a database was established,and statistical software was used to summarize and analyze the data.For the statistical method of syndrome and element,refer to the "simplified measurement" method in "Syndromes and Syndrome Differentiation" to distinguish syndrome and element.Each symptom,sign and other condition data of the patient is weighted and summed(including negative value)according to the suggested syndrome elements to determine the total weight of each syndrome element,so as to make a judgment on the syndrome elements.Statistical analysis of the contribution of the symptoms and signs of the patients to each syndrome element was performed,and the corresponding syndrome element was diagnosed according to the corresponding syndrome element diagnostic criteria.Then,the frequency and frequency of the diagnosed syndrome element were statistically analyzed in SPSS 22 statistical software.Results1.The age distribution of the included patients is 50-59 years old and 60-69 years old,and the proportion of patients with no smoking history is as high as 97.5%.Patients with family history accounted for 19.2% of the total number,and the types were lung cancer,gastric cancer,esophageal cancer,and colorectal cancer in descending order.Most of the patients at the age of menopause were in the age group of 50 to 53 years old,accounting for 49.2%,and the proportion of those who had not yet reached menopause was relatively small.Among the TNM stages,stage I and stage IV were the most common,accounting for 35.8% and 31.7% of the total population,respectively.2.In female lung adenocarcinoma patients,the main disease site syndromes are the lung,followed by the spleen and kidney;among the diseased syndromes,Qi deficiency has the highest frequency,followed by phlegm,yang deficiency,dampness,heat(fire),blood stasis,The occurrence of blood deficiency syndrome elements is relatively infrequent.3.The combination of disease-position syndrome elements is more common in two syndrome elements,accounting for 46.7%.In the combination form of disease syndrome elements,the frequency of single disease syndrome elements is phlegm,qi deficiency,yang deficiency,drink,and qi stagnation;There are relatively more combinations of two or two;the distribution of three or more syndromes is more complicated.4.In the relationship between age and disease-related syndromes,the disease-related syndromes of patients ≤49 years old are mainly phlegm,heat(fire),and qi stagnation;the main disease-related syndromes of 50-59 and 60-69-year-old age groups are both Qi deficiency,phlegm,and yang deficiency;patients aged ≥ 70 years have relatively scattered distribution of syndrome elements.5.In terms of clinical staging,the disease syndromes of stage I patients are mostly phlegm and dampness,followed by qi deficiency,yang deficiency,heat(fire),and qi stagnation;the common symptoms in stage II patients are phlegm,qi deficiency,drink,and qi stagnation;Stage III patients with qi deficiency,yang deficiency,dampness,and drink are more evenly distributed,while stage IV patients are more common with qi deficiency,yang deficiency,phlegm,heat(fire),and yin deficiency.6.Among the correlations between female sex hormone levels and disease-related syndrome elements,the estrogen level of patients with disease-related syndrome elements-yang deficiency was significantly lower than that of other common syndrome elements,and the difference was statistically significant(P<0.05),while testosterone level has no statistical difference with other syndrome elements.ConclutionsThe high incidence of female lung adenocarcinoma is between 50 and 69 years old,and most of them are postmenopausal.The syndrome elements of the patient’s disease location are mostly in the lung,spleen,and kidney;the syndrome elements of the disease are mostly qi deficiency,phlegm,yang deficiency,dampness,and heat(fire).Deficiency factor performance.The pathogenic syndrome elements in the high-incidence age stage are mainly Qi deficiency,phlegm,and Yang deficiency.The middle and early stages of clinical staging are mostly evidences such as phlegm-dampness,while the middle-late syndrome elements are manifested as a mixture of deficiency and excess,such as qi deficiency,yang deficiency,phlegm,dampness,and heat(fire).The estrogen level of the patients with yang deficiency of the disease syndrome element is lower than that of other common syndrome elements,and there is no significant difference in the testosterone content.Part Ⅱ Experimental ResearchObject1.To observe the effect of Yanghe Decoction-modified medicated serum on the proliferation,apoptosis,migration and invasion of human lung adenocarcinoma cells.2.To study the molecular regulation mechanism of Yanghe decoction modified medicated serum interfering with human lung adenocarcinoma cells.3.To observe the effect of Yanghe Decoction and Jiawei Recipe on the growth of lung adenocarcinoma xenografts in nude mice and to verify its mechanism.Content and Method1.Obtain human lung adenocarcinoma A549,H1975 cell lines,prepare Yanghe decoction-containing serum,set up blank group,Yanghe decoction low-dose,medium-dose and high-dose groups,and detect cell growth and cell growth in each group by CCK8 and plate cloning experiments.Proliferation ability,flow cytometry to detect cell apoptosis,scratch and transwell experiments to evaluate cell migration and invasion ability.2.Combined with network pharmacology,predict the molecular regulatory targets and pathways of Yanghe decoction and Jiawei formula intervening in lung adenocarcinoma,The mRNA and protein expression levels of key factors miR-1-3p,PI3 K,AKT and m TOR in lung adenocarcinoma cell lines were detected by q PCR and Western blot.3.Lentiviruses with miR-1-3p overexpression and knockdown were used to transfect lung adenocarcinoma cells(A549,H1975),and q PCR was used to confirm the transfection efficiency,and to establish overexpression and knockdown stable transfectants.The expression changes of PI3 K,AKT and m TOR proteins in the predicted pathway of miR-1-3p overexpressed and non-overexpressed cell lines were detected by Western blot.In order to confirm whether miR-1-3p can compensate for the regulatory effect of Yanghe Decoction medicated serum on lung adenocarcinoma,a rescue experiment was used to detect whether miR-1-3p knockdown could reverse the effect of Yanghe Decoction medicated serum on lung adenocarcinoma.cell effects.4.In vivo experiment,female nude mice were given bilateral ovariectomy + lung adenocarcinoma H1975 cell line in situ to establish a kidney-deficiency lung adenocarcinoma xenograft animal model.Blank group,kidney deficiency group,disease model group,kidney deficiency syndrome group,and Yanghe decoction-modified group were established.Body weight and tumor growth curves were drawn.Western blot and q PCR were used to detect the m RNA and protein expression levels of miR-1-3p,PI3 K,AKT,and m TOR in tumor tissues of each group.Pathological detection and immunohistochemistry were used to evaluate the differences between tumor tissues.Results1.Yanghe Decoction medicated serum inhibits the growth and proliferation of lung adenocarcinoma cells;promotes the apoptosis of lung adenocarcinoma cells,and can regulate the expression of apoptosis-related proteins;at the same time,it can reduce the migration and invasion ability of lung adenocarcinoma cells.2.The molecular regulation prediction results showed that both Yanghe Decoction and miR-1-3p could regulate the PI3K/AKT/m TOR signaling pathway.Experiments showed that Yanghe Decoction-modified medicated serum could increase the expression level of miR-1-3p in lung adenocarcinoma cell lines,and reduce the m RNA and protein expression of PI3K/AKT/m TOR signaling pathway in lung adenocarcinoma cells.The down-regulation of miR-1-3p can block the effect of Yanghe Decoctioncontaining medicated serum on the proliferation,apoptosis and invasion of lung adenocarcinoma cancer cells,and reverse the expression of pathway proteins.miR-1-3p inhibits PI3K/AKT/m TOR signaling pathway in lung adenocarcinoma cells.3.In vivo experiments showed that the growth of tumor tissue in the kidney deficiency syndrome group were more obvious than those in the disease group.Under the HE staining microscope,compared with the disease model group and the kidney deficiency syndrome group,the cancer cells in the Yanghe decoction group had indifferent cytoplasm,rare mitoses,and fibrinoid necrosis.The immunohistochemical results showed that the disease model group The Ki67 protein was highly expressed in the kidney deficiency syndrome group,and the positive rate of Ki67 after the intervention of Yanghe decoction was significantly lower than the former two groups;TUNEL staining showed a small amount of apoptotic cells in the disease model and the syndrome group,and the Yanghe decoction intervention group Apoptotic cells were significantly increased.4.Comparison of serum levels of ALT,AST,BUN and Cr in nude mice in blank group,kidney deficiency syndrome group and Yanghe decoction group showed that each group was in the normal range,and there was no obvious abnormality in liver and kidney function in nude mice after treatment.The content of E2 in the kidney deficiency syndrome group was lower than that in the disease group,while the E2 content in the kidney deficiency syndrome group was significantly increased after the intervention of Yanghe decoction.5.The expression level of miR-1-3p in the disease group was higher than that in the kidney deficiency syndrome group,while the expression levels of PI3 K,AKT,and m TOR were lower than those in the kidney deficiency syndrome group;Syndrome group was significantly increased,and the expression of pathway-related proteins was significantly decreased.ConclusionIn vitro experiments,Yanghe Decoction medicated serum can inhibit the growth and proliferation of lung adenocarcinoma cells by regulating miR-1-3p and then affect the downstream PI3K/AKT/m TOR signaling pathway to inhibit the growth and proliferation of lung adenocarcinoma cells,and promote the apoptosis of lung adenocarcinoma cells.Reduce the migration and invasion ability of lung adenocarcinoma cells.In vivo experiment,the tumor tissue in the kidney deficiency syndrome group were more obvious than those in the disease group,and the m RNA and protein expression levels detected by q PCR and Western blot were statistically different between the two groups,suggesting that kidney deficiency aggravated lung adenocarcinoma to a certain extent.Yanghe Decoction Jiawei Recipe can inhibit the growth of lung adenocarcinoma xenografts in female nude mice with kidney deficiency,reduce the expression of the proliferation index Ki67 protein,promote tumor cell apoptosis,and improve the level of E2 in female nude mice with kidney deficiency.The same detection of regulatory mechanism-related factors showed that in tumor tissue it inhibited the activation ofPI3K/AKT/m TOR pathway by increasing the expression of miR-1-3p.Combining the clinical and experimental research of this topic,in female lung adenocarcinoma patients,TCM syndrome elements are often found in qi deficiency,phlegm,yang deficiency,and dampness.It can be seen that the Yanghe Decoction Jiawei Recipe under the guidance of the treatment of invigorating the kidney,warming yang,nourishing qi and removing phlegm and dampness can inhibit the occurrence and development of female lung adenocarcinoma,and its mechanism is related to the regulation of miR-1-3p and then affect PI3K/AKT /m TOR signaling pathway.