| Polycystic ovary syndrome(PCOS)is one of the most prevalent endocrine disorders in women,which is one of the hottest research topics.Gui Zhu Yi Kun formula(GZYKF)is the empirical formula in treating PCOS.It has been reported that treatment of PCOS using GZYKF is effective.The decrease of ovarian granulosa cells(GCs)in rats with PCOS may be associated with autophagic cell death.GZYKF can mediate the expression levels of Beclin-land LC3,which are related to autophagy.P53/AMPK pathway is the essential pathway to mediate autophagy.The research includes experimental study and clinical study.The aim of the experimental study is to investigate the molecular mechanism associated with GZYKF in the treatment of PCOS by P53/AMPK pathway.The aim of the clinical study is to explore the diagnostic criteria of syndrome factors of PCOS,and to provide an alternative to establish PCOS clinical differentiation criterion.Experimental researchObjective:To investigate the molecular mechanism associated with the traditional Chinese Medicine formula Gui Zhu Yi Kun formula(GZYKF),in the treatment of polycystic ovary syndrome(PCOS).Methods:Firstly,PCOS rat granulosa cells(GCs)were cultured and treated with testosterone propionate(TP)and C2-ceramide for 24h,to establish PCOS cell model in vitro.Secondly,GCs were divided into 6 groups:control group,model group,GZYKF high,middle and low dose groups and 3MA group.Thirdly,western blot was used to observe the expression levels of LC3,P53,P-mTOR and P-AMPK.Lastly,the expression levels of P53,mTOR,AMPK genes were observed by Real-time PCR.Results:Compared with control group,levels of LC3 II was significantly increased in model group(P<0.01).In addition,GCs treated with high dose GZYKF expressed an increase in P53 and P-mTOR(P<0.01,P<0.05),but a decrease P-AMPK expression level was observed(P<0.01).Compared with model group,GCs treated with high dose and middle dose GZYKF expressed an increase in P53 and P-mTOR(P<0.01),but a decrease P-AMPK expression level was observed(P<0.01).Compared with model group,GCs treated with high dose and middle dose GZYKF expressed an increase in P53 and mTOR genes(P<0.01),but a decrease AMPK gene was observed(P<0.01).Conclusion:PCOS cell model in vitro is established.The effects of GZYKF in mediating the p53/AMPK pathway may inhibit GC autophagy,which suggests a possible novel mechanism underlying the treatment of PCOS with GZYKF.Clinical researchObjective:To explore the diagnostic criteria of syndrome factors of PCOS,and to provide an alternative to establish PCOS clinical differentiation criterion.Methods:PCOS information Collection Table was created.The medical history,clinical symptoms,signs and physical chemical index of 518 PCOS patients participated in this clinical study were collected,using the cross-sectional study method.Epidata3.1 database was established.Latent variable analysis was used to create TCM syndrome factor diagnosis criteria of PCOS.After which,the diagnosis that the model derived was compared with the clinical diagnosis.Results:1.Both goodness of fit index(GFI)and GFI adjusted for degrees of freedom approximated 1,suggested that fitting model was good.RMSEA Estimate was 0.0637≤0.08,which indicated that the model was acceptable.2.The results of the consistent state analysis were below.Kidney,liver or spleen,only having one symptom in model could be diagnosed.And the rates of the correct diagnosis were 71.6%,88%and77%respectively.Having one of the three symptoms,lumbosacral pain,frequent urination at night and tinnitus,could diagnose kidney.Having one of the three symptoms,depression,impatience and preference for sighing,could diagnose liver.Having one of the three symptoms,poor appetite,abdominal distention and diarrhea,could diagnose spleen.Having two symptoms in model could be diagnosed qi deficiency,qi stagnation or phlegm-damp.And the rates of the correct diagnosis were 86.3%,83.2%and 80.5%respectively.Having two of the three symptoms,weakness,fatigue and spontaneous perspiration,could diagnose qi deficiency.Having two of the three symptoms,chest tightness,chest pain and breast pain,could diagnose qi stagnation.Having two of the three symptoms,sleepiness,head as wrapped and phlegm,could diagnose phlegm-damp.When dysmenorrhea,blood clots and dark complexion were all seen,blood stasis could be diagnosed and the rate of correct diagnosis was 51.7%.3.The most 3 common syndrome factors of disease location in PCOS were kidney,liver and spleen,the frequency of them were 332,317 and 195 respectively.The most 5 common syndrome factors of disease characters in PCOS were blood stasis,qi deficiency,qi stagnation,phlegm-damp and yang deficiency,the frequency of them were261,206,114,108,and 103 respectively.4.The most 4 common syndrome types were kidney deficiency and blood stasis syndrome,kidney qi deficiency,liver depression and spleen deficiency,and spleen qi deficiency,and the frequency of them were 34.75%,25.10%,24.90%and 20.8%respectively.5.The frequency of kidney significantly increased in infertility group,when compared with fertile group(P<0.01).The frequency of blood stasis,phlegm-damp and qi stagnation,significantly increased in obesity group,when compared with non-obesity group(P<0.05).The frequency of spleen and phlegm-damp significantly increased in hyperandrogenism group,when compared with no hyperandrogenism group(P<0.01).The frequency of fire heat significantly increased in LH/FSH>2 group,when compared with LH/FSH≤2 group(P<0.01).The frequency of kidney,phlegm-damp and qi deficiency,significantly increased in insulin resistance group,when compared with no insulin resistance group(P<0.05).6.The most 2 common NIH types of PCOS were NIH Ⅰ and NIHⅣ,and the frequency of them were 50.19%and 42.66%respectively.However,the distribution of syndrome factors of the two types were the same(P>0.05).Conclusion:1.The diagnostic criteria of syndrome factors of PCOS is established.2.The most 3 common syndrome factors of disease location of PCOS were kidney,liver and spleen.The most 5 common syndrome factors of disease characters of PCOS were blood stasis,qi deficiency,qi stagnation,phlegm-damp and yang deficiency.3.The most 4 common syndrome types were kidney deficiency and blood stasis syndrome,kidney qi deficiency,liver depression and spleen deficiency and spleen qi deficiency.4.Infertility issues are closely related with Kidney.Obesity relates to blood stasis,phlegm-damp and qi stagnation.Hyperandrogenism relates to spleen and phlegm-damp.The increase of LH relates to fire heat.Insulin resistance relates to kidney,phlegm-damp and qi deficiency.5.The most 2 common NIH types of PCOS were NIH Ⅰ and NIHⅣ,and the distribution of syndrome factors of the two types were the same. |
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