Study On The Correlation Between Clinical Characteristics And TCM Syndromes Of PCOS And The Mechanism Of Modified Longdan Xiegan Decoction In Treating PCOS

BackgroundPolycystic ovary syndrome（PCOS）is a common endocrine disorder irn reproductive women.It is refered to the category of delayed menstrual cycle,amenorrhoea,metrorrhagia,infertility,and other disease syndromes of traditional Chinese medicine（TCM）,Patients with PCOS often suffer from complications affecting their reproductive health and quality of life,and their disordered endocrine status and metabolic environment increase their risk of other long-term complications.It has become a hot and difficult point in medical research.The long-term effect of traditional Chinese medicine in treating this disease is better than that of western medicine.At present,the treatment of polycystic ovary syndrome with traditional Chinese medicine is mainly based on the combination of TCM syndrome differentiation and disease differentiation,and has achieved good results.The modified Longdan Xiegan Decoction is a clinical prescription summarized by Professor Yu-zhen Zhang,a TCM gynecologist at the First Affiliated Hospital of Guangzhou University of TCM.It is an effective prescription for the treatment of liver-induced stagnation of PCOS.Therefore,this study is based on the Modified Longdan Xiegan Decoction on the basis of PCOS,focusing on the changes of hormone levels,inflammation levels,and the mechanism of signaling pathways.This study may provide valuable insights into the value advantages of TCM prescriptions and diagnosis and treatment methods for PCOS treatment.Furthermore,the study may promote fUrther investigation on the moderm pharmacological effects of the modified Longdan Xiegan Decoction.Objective1.Clinical research:General data were collected from patients with PCOS.The distribution of TCM syndrome types in the patients,the distribution pattern of Western medical phenotypes,and the correlation between syndrome types and phenotypes were analyzed.The sex hormone levels,glucose metabolism indicators,and chronic inflammatory factors of the patients were obtained,and the relationships among sex hormone level,glucose metabolism index,chronic inflammatory factor,PCOS TCM syndrome type,and Western medicine phenotype were determined.Thus,our aims were to provide a basis for the clinical treatment,disease classification,disease causes,and physiological symptoms of PCOS and to deepen the awareness of clinical workers about this disease in order to guide clinical diagnosis and prescription.2.We performed an experimental study that replicated PCOS model rats by the poresky method to investigate the pharmacodynamic effects and mechanism of the modified Longdan Xiegan Decoction on the PCOS model.The effect of the Modified Longdan Xiegan Decoction on PCOS was confirmed.Based on the theory of inflammation,the prescription reduces inflammation,restores glucose metabolism to normal levels,and regulates hormone levels and the related inflammation JAK2/STAT3 signaling pathway.This study is eonducive to promoting the modern drug value of the Longdan Xiegan Decoction in the appli’cation and development of classic prescriptions for the "stagnant fire in Gan channel type" disease.Method1.Of the 136 patients enrolled in the study,102 were included in the PCOS group;and 34-who were women without any disease,in the control group.General Case situation:name,age,history of marriage and childbirth,menstrual history,matermal history,family history,and visiting a doctor.Physical examination:height,weight,waist circumference,hip circumference;calculated BMI=weight（kg）/height2（m2）and waist-to-hip ratio（WHR）.Combine the tongue and pulse to determine the type of TCM Syndrome.Sex hormones and gonadotropin-releasing hormone:chemiluminescence detection of serum follicle stimulating hormone（FSH）,luteinizing hormone（LH）,testosterone（T）levels,and ELASA for serum leptin and kisspeptin.Biochemical tests:automatic biochemical analyzer to determine fasting blood glucose（FPG）,radioimmunoassay deternination of fasting insulin（FINS）levels,and IL-4,IL-6,IL-8,IL-10 by the ELASA.Phenotypic classification and correlation were analyses of patients with PCOS.2.Establishment rat PCOS model by insulin（INS）combined with human chorionic gonadotropin（HCG）.The experiment was set up with control,model,Da Ying-35（0.21 g/kg）,and modified Longdan Xiegan Decoction（29.6 g/kg）/low-dose group（7.4 g/kg）5 continuous intragastric administration for 15 days;the weights and sizes of the ovaries were measured,and changes in the weights of animals treated by Modified Longdan Xiegan Decoction were monitored.Comparison the luteinizing hormone,follicle stimulating hormone,testosterone,fasting blood glucose,fasting insulin hormone levels and IL-2,IL-4,IL-6,IL-10 inflammatory factors in each group.WB was detection of each group of ovarian JAK2,STAT3,p-STAT3,and IL-6 expression levels.Results1.Results of Clinical study:（1）.The characteristics of distribution:A total of 102 patients with PCOS were enrolled in this study.The phenotypic distribution:Classical type in 31 cases（30%）,hyperandrogenic anovulatory type in 12 cases（12%）,hyperandrogenic ovulatory type in 8 cases（8%）,and non-hyperandrogenic type in 51 cases（50%）.Syndrome distribution:kidney deficiency type in 27 cases（26.5%）,stagnation fire in Gan channel type in 38 cases（37.3%）,spleen deficiency and dampness type in 18 cases（17.6%）,Qi stagnation and blood stasis type in 19 cases（18.6%）.（2）.The levels of Sex hormones:phenotypes Compared:The LH,T level and LH/FSH ratio of the Classical type patients were the highest,and the difference was statistically significant（P<0.05）.The T levels from the largest to the smallest were classical type>hyperandrogenic ovulatory type>hyperandrogenic anovulatory type>non-hyperandrogenic type,and the difference between the classical type T level and non-hyperandrogenic type and hyperandrogenic anovulatory type was statistically significant（P<0.05）.Syndrome distribution:the LH,T level and LH/FSH ratio of stagnation fire in Gan channel type are the highest,and the T level is from high to low:stagnation fire in Gan channel type>spleen deficiency and dampness type>Qi stagnation and blood stasis type>Kidney deficiency type,and the level of stagnation fire in Gan channel type T and other syndromes were statistically different（P<0.05）.（3）.The characteristics of glucose metabolism:phenotypes Compared:Fasting insulin was higher in all PCOS patients than in the normal group（P<0.05）,and there was no statistically significant difference between the patients（P>0.05）.The HOMA-IR of all PCOS patients was higher than that of the normal group（P<0.05）,but there was no significant difference between the patients（P>0.05）.Syndrome types compared:HOMA-IR high to low order:spleen deficiency and dampness type>stagnation fire in Gan channel type>Qi stagnation and blood stasis type>kidney deficiency type,and compared with kidney deficiency type,HOMA-IR difference of spleen deficiency and dampness type Statistically significant（P<0.05）.（4）Serum Leptin and kisspeptin levels:compared with each phenotype:serum Leptin and kisspeptin levels were higher in the PCOS patients than in the normal group.The difference between non-hyperandrogenic type and the control group was statistically significant（P<0.05）,and syndrome types compared:spleen deficiency and dampness type of Leptin is the largest,which is higher than the other three syndrome types.The order of Leptin is as follows:spleen deficiency and dampness type>stagnation fire in Gan channel type>kidney deficiency type>Qi stagnation and blood stasis type.（5）Characteristics of inflamnatory factors IL-4,IL-6,IL-8,IL-10:Phenotypes compared:IL-6 and IL-8 inflammatory factors in PCOS patients were higher than those in the control group,and the differences were Statistically significant（P<0.05）,Among them,non-hyperandrogenic type patients had the highest IL-6 levels,The levels of IL-4 and IL-10 were lower in the control group than those in the control group,and the levels of IL-4 and IL-10 in the non-hyperandrogenic type PCOS patients were significantly different（P<0.05）.Syndrome types compared:the level of IL-6 is as follows:stagnation fire in Gan channel type>spleen deficiency phlegm type>kidney deficiency type>Qi stagnation and blood stasis type;IL-4 high to low order is;stagnation fire in Gan channel type>Qi stagnation and blood stasis type>kidney deficiency type>spleen deficiency and dampness type.2.Experimental research results:（1）Evaluation of PCOS rat model:Insulin（INS）combined with human chorionic gonadotropin（HCG）method to replicate rat PCOS model rat vaginal exfoliated cell smear without estrus cycle change suggesting no ovulation and showing weight gain,HE pathological biopsy The polycystic changes of the ovary and the increase of serum testosterone（T）in the model group were significantly different from those in the normal control group（P<0.05）,suggesting that the model was successful and met the diagnostic criteria of PCOS patients.（2）Indicators after drug intervention:①Comparison of body weight:After 14 days of treatment with Modified Longdan Xiegan Decoction,it has obvious therapeutic effect on PCOS model rats,which can reduce the body weight,ovarian weight and ovarian volume of model animals（P<0.05）and improve the pathological morphology of ovarian tissue.②The levels of Sex hormones:The serum T and LH levels in the model group were higher than those in the normal control group.Compared with the model group,the addition and subtraction of Modified Longdan Xiegan Decoction significantly decreased serum luteinizing hormone（LH）and testosterone（T）in PCOS rats,and insulin（INS）levels（P<0.05/P<0.01）.③The levels of serum IL-2 and IL-6 in the model group were higher than those in the normal control group.IL-4 and IL-10 were lower than the normal control group.Compared with the model group,the addition and subtraction of Modified Longdan Xiegan Decoction increased after treatment.Inflammatory factors IL₄,IL-10（P<0.05）and inhibition of proinflammatory cytokines IL-2,IL-6 levels（P<0.05）;WB analysis of ovarian tissue,compared with the normal control group,the expression of JAK2 and STAT3 protein in the model group increased,the expression of p-STAT3 and IL-6 protein decreased in each dose group of Modified Longdan Xiegan Decoction,confirming the addition and subtraction of gentian Xiexan Decoction can increase JAK2,STAT3 and decrease the expression of p-STAT3 and IL-6.④Serum Leptin and kisspeptin levels:The serum levels of Leptin and kisspeptin in the model group were higher than those in the normal control group.Compared with the model group,the levels of Leptin and kisspeptin decreased after the intervention of each dose group of Modified Longdan Xiegan Decoction,and the high dose group decreased.Most obvious,but not statistically different from the positive drug group.Conclusion1.Clinical research:①1.50%of patients with PCOS show hyperandrogenemic type,and the T-level of patients with classic type is significantly higher.PCOS patients with Chinese and Western phenotype non-hyperandrogenemic type patients have more severe insulin resistance and light glucose damage in hyperandrogen anovulatory patients.TCM is common in patients with stagnant fire in Gan channel type and kidney deficiency type,followed by spleen deficiency and dampness type and Qi Stagnation and Blood Stasis type.In patients with classic PCOS,there were more patients with stagnant fire in Gan charnel syndrome,while non-hyperandrogenemic PCOS patients had more kidney deficiency syndrome.②The serum inflammatory factors of PCOS patients with different Western medical phenotypes and TCM syndromes were higher than those of the control group,and the level of pro-inflammatory factor IL-6 was significantly increased.The level of IL-10 was lower than that of the control group,suggesting that PCOS patients are chronic inflammation status.The sex hormones in patients with PCOS are directly related to their characteristic clinical phenotypes,and the high T level is related to the phenotype of liver stagnation syndrome.High glucose and high Leptin is associated with spleen deficiency and dampness syndrome phenotype,while inflammatory factors are associated with various phenotypes of traditional Chinese medicine,and they may play a key role in the pathogenesis of the disease.2.Experimental Study:①Insulin（INS）combined with human chorionic gonadotropin（HCG）replicates the rat PCOS model.Its morphological changes and endocrine changes in ovary are similar to those of clinical PCOS patients.It is an ideal PCOS animal model.②Modified Longdan Xiegan Decoction can reduce the body weight,ovarian weight and ovarian volume of PCOS model rats,significantly reduce serum LH,T and insulin levels in PCOS rats,and decrease serum Leptin and kisspeptin levels.③After treatment with Modified Longdan Xiegan Decoction,the levels of anti-inflammatory factors IL-4 and IL-10（P<0.05）and the levels of pro-inflammatory cytokines IL-2 and IL-6 were increased,and the mechanism may be related to the activation of JAK2/STAT3 pathway.The effect is related.