| BackgroundAlthough it is related to the dysfunction of the HPO axis and the use of sex hormones is the first-line treatment for hemostasis,there are also contraindations for its use.In addition,abnormal local endometrial coagulation function is also an important pathogenesis,so the use of hemostasis drugs is also very important.Er Nian Decoction is an experienced prescription for the treatment of metrorrhagia and metrostaxis made up by Professor Luo Yuankai,a master of traditional Chinese medicine and the founder of Lingnan Luoshi Gynecology.Preliminary clinical studies have shown that Er Nian Decoction can effectively stop bleeding in the treatment of metrorrhagia and metrostaxis,and the systemic symptoms are well improved without obvious adverse reactions,but there is a lack of high-level evidence-based medical research.Previous animal experimental studies have shown that Er Nian Decoction can shorten the bleeding time of normal mice and heparinized mice,and it is speculated that its hemostasis may be related to the inhibition of tissue-type plasminogen activator(t-PA)activity and the reduction of fibrin degradation,but the relevant pharmacodynamic substance basis and molecular mechanism are unknown.ObjectiveRandomized,positive control and prospective clinical study method was used to observe the clinical efficacy and safety of Er Nian Decoction in the treatment of spleen-kidney uterine bleeding.Through network pharmacological method,the possible pharmacodynamic substance basis and related mechanism of action of Er Nian Decoction in the treatment of metrorrhagia and metrostaxis were further discussed,which laid a foundation for the further development of Lingnan characteristic new drugs.Methods1.Clinical Research:Noninferiority trial and randomized,controlled and prospective study methods were adopted.A total of 82 patients who met the diagnostic criteria for splenic-kidney deficiency and leakage admitted to the First Affiliated Hospital of Guangzhou University of Chinese Medicine from October 2019 to March 2021 were collected.Finally,40 patients were enrolled and randomly divided into the Er Nian Decoction observation group and the adrenal color hydrazone tablet control group,with 20 cases in each group.Hemostasis time was taken as the main efficacy index,and duration of hemostasis,TCM syndrome score,and intima thickness before and after treatment were taken as secondary efficacy indexes.SPSS26.0 software was used for statistical analysis.Measurement data were expressed as mean ± standard deviation(x±s)by T test,and counting data(rate)was tested by chi-square test.To evaluate the clinical effect of Erlin-decoction on hemostasis of spleen-kidney deficiency.To evaluate the clinical safety of Er Nian Decoction,the vital signs,blood analysis,coagulation,liver function,serum creatinine indexes and adverse reactions of the subjects before and after treatment were taken as safety indexes.2.Network Pharmacological Research : Through network pharmacological techniques,the effective active ingredients were obtained by searching in TCMSP database and TCMID database,with the combination of molecular drug DL,oral bioavailability OB,Lipinski rule and Veber rule.TCMSP and Swiss Target Prediction database were used to predict the corresponding target of active ingredients.The related target of disease was obtained by searching for ‘ovarian abnormal uterine bleeding’in Gene Cards and OMIM database.And the co-target gene can be obtained through the use of gene mapping in Venn diagram.The protein-protein interaction(PPI)network was constructed by STRING database,the biological function annotation of GO was carried out by Metascape,the key genes in PPI and kyoto encyclopedia of genes and genome(KEGG)pathway enrichment were carried out by R language,and the component-target network was constructed by Cytoscape 3.7.2 software.Molecular docking carried out with Py Mol and Auto Dock Tool software.By screening the active components of Er Nian Decoction,the potential targets and related signaling pathways of Er Nian Decoction were predicted,and the molecular mechanism of Er Nian Decoction in the treatment of ovulatory disorders with abnormal uterine bleeding was preliminarily discussed.Results1.Clinical Research:82 cases of spleen-kidney deficiency were screened,42 cases were excluded and 40 cases were included,20 cases were in the Er Nian Decoction observation group and 20 cases were in the Carbazochrome Tablets control group.In the traditional Chinese medicine group,1 case fell off(stopped medication by itself and did not complete the course of medication),and in the western medicine group,2 cases stopped the test(1 case had adverse reaction vomiting and 1 case had aggravated bleeding symptoms).Finally,19 cases in the Er Nian Decoction observation group and 18 cases in the Carbazochrome Tablets control group were collected.(1)General Information Before Treatment:In the Er Nian decoction observation group,the oldest age was 53 years old,the youngest was 16 years old,and the average age was33.95±10.51 years old.There were 3 patients aged 13-20 years old,13 patients aged 21-45 years old,and 3 patients aged 46-55 years old.In the control group,the oldest age was 54 years old,the youngest was 23 years old,the mean age was 35.00±10.48 years old,13 cases were 21-45 years old,and 5 cases were 46-55 years old.There was no significant difference in the mean age and age distribution between the two groups before treatment(P>0.05),which showed comparability.The average number of births was 1.00±1.05 in the observation group and 1.06±1.00 in the control group.In the observation group,the maximum BMI was 28.04,the minimum BMI was 16.8,and the mean BMI was20.71±2.90;in the control group,the maximum BMI was 30.9,and the minimum BMI was18.5,and the mean BMI was 21.65±3.28.Before treatment,11 cases were mild and 8 cases were moderate in the observation group,with an average score of 12.42±4.10.In the control group,13 cases were mild and 5 cases were moderate,with an average score of12.11±4.64.There was no statistical significance in the average number of births,BMI and severity of disease between the two groups before treatment(P>0.05),which showed clinical comparability.(2)Clinical Data Before Treatment:The total clinical symptom score of Er Nian decoction observation group was 12.42±4.10 points,the primary symptom score was8.53±3.26 points,and the secondary symptom score was 3.89±2.89 points.The total clinical symptom score of Carbazochrome Tablets control group was 12.11±4.64 points,the primary symptom score was 8.44±2.96 points,and the secondary symptom score was3.67±2.74 points.There was no significant difference in total score,primary symptom score and secondary symptom score between the two groups(P>0.05).Before treatment,the average hemoglobin of the observation group was 123.84±17.29g/L,and that of the control group was 130.67±12.41g/L.There was no significant difference in hemoglobin between the two groups before treatment(P>0.05).The mean endometrial thickness before treatment was 7.77±3.70 mm in the observation group and 8.42±3.40 mm in the control group.There was no significant difference in endometrial thickness between the two groups before treatment(P>0.05).Before treatment,there were no significant differences in all safety indexes(WBC,PLT,PT,APTT,FIB,SCR,AST,ALT)between two groups(P>0.05).All clinical data of the two groups were comparable before treatment.(3)Hemostatic Efficacy: After treatment,13 cases in Er Nian decoction observation group had hemostasis,of which 4 cases had hemostasis time ≤3 days,9 cases had hemostasis time ≤ 4-7 days,the average hemostasis time was 4.62±1.90 days,and 9 cases in Carbazochrome Tablets control group had hemostasis time ≤3 days,1 case had hemostasis time ≤3 days,and 8 cases had hemostasis time ≤ 4-7 days,the average hemostasis time was 5.33±1.58 days.The hemostasis rate of Er Nian decoction group(68.4%)was higher than that of Carbazochrome Tablets(50.0%),and the average hemostasis time of Er Nian decoction group was lower than that of control group,and there was no significant difference between the two groups(P>0.05).The average maintenance time of hemostasis in the observation group was 21.15±7.50 days,and the average maintenance time of hemostasis in the control group was 17.22±5.17 days.Although the average maintenance time of hemostasis in the Er Nian decoction group was higher than that in the control group,there was no statistical significance between the two groups(P>0.05).The average duration of hemostasis was 9.33±2.52 days in the observation group(3 cases)and 12.50±3.00 days in the control group(4 cases).There was no significant difference between the two groups(P>0.05).The average duration of hemostasis was24.70±3.62 days in the observation group(10 cases)and 21.00±2.55 days in the control group(5 cases).There was no significant difference between the two groups(P>0.05).Observation group recently hemostatic efficacy available(3 days)or less 4 cases,9 cases,6 cases ineffective,the total effective rate was 68.4%,the control group in the near future hemostatic efficacy available in 1 case,8 cases effectively,invalid 9 cases,the total effective rate was 50.0%,Er Nian decoction group hemostatic total effective rate is higher than that of Carbazochrome Tablets group,but there was no statistically significant difference is compared between two groups(P>0.05).There was no significant difference between the two groups in different age,different BMI,different bleeding time and comparison of hemostatic efficacy(P>0.05).(4)Syndrome Curative Effect:In the integral comparison of TCM clinical syndrome efficacy,the total effective rate of the observation group was 68.4%,and the total effective rate of the control group was 50.0%,the difference between the two groups was statistically significant(P<0.05).Within the two groups of traditional Chinese medicine clinical score comparison results show that Er Nian decoction group before and after treatment in improving main symptoms and secondary symptoms difference was statistically significant(P<0.05),Carbazochrome Tablets group before and after treatment in improving main symptoms difference was statistically significant(P<0.05),but there was no statistically significant difference in improving secondary symptom(P>0.05).After treatment,there were statistically significant differences in the total symptom score and secondary symptom score between the two groups(P<0.05),while there was no statistically significant difference in the main symptom score(P>0.05).After treatment,Er Nian decoction was superior to Carbazochrome Tablets group in improving fatigue,waist and knee pain and weakness,chills and cold limbs,and abnormal complexion,with statistical significance(P<0.05),while there was no statistical significance in dizziness and poor diet(P>0.05).(5)Comparison of Clinical Data:After treatment,the average hemoglobin in the Er Nian decoction group was 119.79±17.58g/L,and that in the Carbazochrome Tablets group was 127.56±12.59g/L.After treatment,the average hemoglobin in the two groups was lower than that before treatment,and the differences between the two groups were statistically significant(P<0.05).There was no significant difference in hemoglobin between the two groups after treatment(P>0.05).Er Nian decoction group after treatment,on average,endometrial thickness was 7.87±3.31 mm,Carbazochrome Tablets group after treatment,on average,endometrial thickness was 7.29±2.55 mm,There was no significant difference in endometrial thickness between the Er Nian decoction group and the Carbazochrome Tablets group(P>0.05).There was no significant difference in endometrial thickness between the two groups after treatment(P>0.05).In Er Nian decoction group,13 cases had hemostasis after treatment,11 cases completed 3-month follow-up,and 6 cases did not relapse after 3 months.After Carbazochrome Tablets groupt treatment,9 cases had hemostasis,8 cases completed 3-month follow-up,4 cases did not relapse after 3months.The recurrence rate of one month in Er Nian decoction group(9.1%)was significantly lower than that in Carbazochrome Tablets group(37.5%),and there was no statistical difference in the recurrence rate between the two groups(P>0.05).The recurrence rate of two months in Er Nian decoction group(27.3%)was lower than that in Carbazochrome Tablets group(50.0%),and there was no statistical difference in the recurrence rate between the two groups(P>0.05).At three months,the recurrence rate of Er Nian decoction group(45.5%)was lower than that of Carbazochrome Tablets group(50.0%),and there was no statistical difference in the recurrence rate between the two groups(P>0.05).(6)Safety comparison:There were no significant differences in safety indexes(WBC,PLT,PT,APTT,FIB,SCR,AST,ALT)between the two groups before and after treatment(P>0.05).There were no significant differences in the safety indexes between the two groups after treatment(P>0.05).(7)Adverse Reactions:One case of Carbazochrome Tablets group showed vomiting,while Er Nian decoction group showed no adverse reaction.There was no statistical significance in the incidence of adverse reactions between the two groups by chi-square test(P=0.298>0.05).2.Network Pharmacological Research : 84 active components and 236 intersection targets of Er Nian decoction were obtained by screening.Among the active ingredients,quercetin,luteolin and naringenin were the top three with degree values.In Er Nian decoction,sovereign drug Minorhizoma contains quercetin,luteolin,naringin three components,sovereign drug Kang Minorhizoma,Fuyao mulberry parasitic contains quercetin,minister medicine Codonopsis contains luteolin,licorice contains naringin.Among the key targets,STAT3,MAPK1,MAPK3,EP300,Akt1 and Jun had higher degree values.KEGG pathway enrichment analysis showed that PI3K-Akt signaling pathway was the most significant.(1)Quercetin: belong to flavonoids,which can reduce the hypothalamus-pituitary-ovarian axis(HPO axis)of estrogen receptor alpha(ER alpha),estrogen receptor beta(ER beta)and progesterone receptor(PR)expression in regulating serum follicle hormone(FSH)and luteinizing hormone(LH)level,promote the recovery of ovarian function and the menstrual cycle.(2)Promote the development and proliferation of follicles and granulosa cells at all levels,and enhance the secretion of estrogen in granulosa cells.(3)The proliferation of human umbilical vein endothelial cells and the ability of angiogenesis in vitro can be inhibited by decreasing the m RNA expression of intracellular vascular endothelial growth factor(VEGF)and basic fibroblast growth factor(b FGF).(4)It can effectively inhibit the proliferation and migration of vascular smooth muscle cells(VSMC)induced by thrombin,inhibit endometrial hyperplasia,and reduce platelet aggregation and release.(5)can reduce the adhesion between platelets and cells,and can interfere with internal/external coagulation and correct coagulation dysfunction.(6)has obvious anticoagulant activity,can significantly reduce the inflammatory cells produced by nitric oxide(NO),tumor necrosis factor-A(TNF-α)and interleukin-6(IL-6),in order to protect vascular endothelial function.(2)Luteolin:(1)has obvious pro-coagulant activity,can inhibit the phosphorylation of nuclear factor kappa B(NF-κB),reduce the production of NO,IL-6 and other inflammatory cells,play an anti-inflammatory effect.(2)can increase the thickness of the endometrium,with the double effects of estrogen-like and anti-estrogen-like.(3)To promote the secretion of estradiol from ovarian granulocyte cells.(4)The expression of VEGF in rabbit corneal vessels and human umbilical vein endothelial cells was significantly inhibited.(3)Naringin:(1)inhibit the inflammatory cell infiltration,the release of inflammatory mediators,the activity of myeloperoxidase(MPO)and inducible nitric oxide synthase(i NOS),inhibit the secretion of TNF-α to achieve anti-inflammatory effect.(2)It can inhibit platelet aggregation in hyperlipidemia rabbits.(3)It can effectively inhibit the platelet aggregation induced by various stimulants(ADP,collagen,thrombin),and has the strongest inhibitory effect on the platelet aggregation induced by ADP.(4)Key targets: The high expression of signal transduction and transcriptional activator 3(STAT3)in endometrial cancer suggests that long-term and repeated abnormal uterine bleeding is prone to endometrial lesions and may be related to the expression of STAT3 gene.STAT3 can accelerate the occurrence and development of tumors by inhibiting apoptosis,inducing cell proliferation,promoting angiogenesis,invasion and metastasis,inducing inflammation and immunosuppression,etc.Mitogen-activated protein kinase(MAPK)is mainly involved in cell growth,development,apoptosis,immune regulation and other cell signaling activities.MAPK is a regulator of angiogenesis induced by fibroblast growth factor 2(FGF2).FGF2 has chemotactic and mitotic effects on endothelial cells,promotes angiogenesis,and leads to angiogenesis.Previous studies have found that Survivin,Bcl-2,Bax and other apoptotic factors are involved in the process of abnormal hyperplasia of AUB endometrium,and it is speculated that STAT3,MAPK1 and MAPK3 may also be involved in the process of apoptosis of AUB endometrium cells,which still needs further study and confirmation.(5)Signaling pathway: the PI3 K signaling pathway is considered to be one of the most important signaling pathways for ADP-induced platelet activation,plays an important role in platelet aggregation and thrombus stabilization,participates in the regulation of VEGF secretion,and completes the control of vascular and vascular endothelial cell apoptosis.(6)Molecular docking: it showed that the main active ingredients had good binding ability to key targets.ConclusionClinical studies : Er Nian Decoction has a clear effect on hemostasis of spleen-kidney deficiency type leakage,and is superior to adrenaline-color hydrazone tablet in improving TCM syndromes,especially in improving the overall symptom score and secondary symptoms,with no adverse reactions.Network pharmacological studies:Quercetin,luteolin,naringin and other compounds may be the main active compounds in the hemostasis and diabetes control of Erminshu decoction,which may play a role in regulating PI3K-Akt signaling pathway by regulating key targets such as STAT3,MAPK1,MAPK3 and so on. |
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