Study On Plasma Metabolomics Of Blood Stasis Due To Cold Syndrome And Intervention Effects Of Shaofu Zhuyu Decoction

The analytical method of plasma metabolomics was established by employing LC-MS technique.Based on blood stasis due to cold syndrome rat model made by ice water bath,the endogenous metabolites that exist difference between model group and control group were analyzed.The possible metabolic pathways related to the syndrome were discussed primarily.The action mechanisms of Shaofu Zhuyu decoction on blood rheological properties and related functions of ovary and uterus were investigated for blood stasis due to cold syndrome model rats.The main contents are summarized as follows.1.Literature investigationThe research progress on preparation and evaluation of animal models of blood stasis due to cold syndrome was systematically overviewed.The experimental research progress of the mechanisms of dysmenorrheal results from blood stasis due to cold syndrome,and the research progress of Shaofu Zhuyu decoction and its action mechanism for curing dysmenorrheal results from blood stasis due to cold syndrome were summarized.The general workflow,analytical techniques,data processing method and the application in the research of traditional Chinese medicine were systematically summarized for metabolomics.2.Plasma metabolomics research for model rats of blood stasis due to cold syndromeBased on the indexes of blood rheology,blood routine and blood clotting,etc.,the ice water bath time was observed for preparing the blood stasis due to cold syndrome rat model.The results showed that the indexes of blood rheology reach the max extent of abnormity with the modeling time of 10 d.The analytical method of plasma metabolomics was established by employing high performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry?HPLC-QTOF MS?.The multi-variant data were recognized of patterns by principal components analysis-discriminant analysis?PCA-DA?and tested of significance by t-test.The metabolic network was constructed by using Ingenuity Pathway Analysis?IPA?,and furthermore,the regularities of metabolism and perturbations for the model rats of blood stasis due to cold syndrome and the changes of plasma metabolic profiling following with the time of ice water bath were discussed As a result,the difference between the control group and the 10 d model group was the most significant,and the 10 d model group could also be separated from the 5 d and 15 d model groups.Furthermore,10 potential biomarkers were identified qualitatively,which belonged to six categories,including amino acids,phosphatidylcholines,arachidonates,phosphatidylcholines,sphingosines and bile acids.Five metabolic pathways were screened primarily as the potential ones for the model of blood stasis due to cold syndrome,i.e.sphingolipid metabolism,glycerophospholipid metabolism,ether lipid metabolism,cysteine and methionine metabolism and primary bile acid biosynthesis.3.The relationship between oxidative stress and blood stasis formationTwo different rat model of blood stasis due to cold syndrome were established by ice water bath and ice water bath combined with injection of adrenalin.respectively.The indexes of serum oxidative stress factors?SOD and MDA?,inflammatory cytokines?IL-6 and TNF-2a?and vasomotor factors?ET-1,NO and NOS?were measured.The relationships between the three factors were analyzed by bivariate correlation.The results showed that whole blood viscosity,plasma viscosity,malondialdehyde?MDA?,cell necrosis factor-2a?TNF-2a?,interleukin-6?IL-6?and endothelin^-1?ET-1?of the two groups of model rats increased,compared with those of the control group,while superoxide dismutase?SOD?,nitric oxide?NO?,nitric oxide synthase?NOS?decreased.Moreover,IL-6 and TNF-2a were positively correlated with MDA and ET-1,and negatively correlated with SOD,NO and NOS;NO and NOS were positively correlated with SOD,and negatively correlated with MDA;ET-1 was negatively correlated with SOD,and positively correlated with MDA.It is suggested that the abnormal changes of oxidative stress reaction should promote the formation of blood stasis.4.Intervention effects study of Shaofu Zhuyu decoction on the model rats of blood stasis due to cold syndromeThe blood stasis due to cold syndrome rat model was made by ice water bath,and Shaofu Zhuyu decoction?SFZYD?was given to intervene.The related indexes were measured,and the relationships were analyzed by bivariate correlation and partial correlation.What's more,the intervention effects on blood rheoloical properties and the relative functions of ovaries and uterus.?1?Effects on blood rheological properties and ovarian secretionThe results showed that,compared with those of the control group,whole blood viscosity,plasma viscosity and content of FIB of the model group increased,while TT and APTT reduced.but PT prolonged.Besides,the content of estrogen?E₂?in serum increased but that of progestogen?P?decreased.It can be observed in SFZYD high-dose group that,compared with those of the model group,whole blood viscosity,plasma viscosity and content of FIB were decreased.TT and APTT prolonged but PT reduced,serum E₂ was decreased and P was increased.Besides,E₂ in serum was observed to be positively correlated with whole blood viscosity(1 s^-1)and plasma viscosity,and negatively correlated with TT and APTT;P in serum was negatively correlated with whole blood viscosity(1 s^-1)and plasma viscosity,and positively correlated with TT and APTT.It is suggested that Shaofu Zhuyu decoction could correct the abnormal blood rheology,restore the disordered coagulation function,regain the blood supply to reproductive organs,promote the ovarian development and regulate the endocrine function to make the secretion of estrogen and progestogen tend to the normal level;the therapeutic effects of high-dose were better than those of low-dose.?2?Effects on ovarian inflammatory cytokines and vasomotor factorsThe results showed that,compared with those of the control group,serum estrogen?E₂?of the model group increased,while progestogen?P?decreased.Besides,ovarian inflammatory cytokines?IL-6 and TNF-2a?and vasomotor factors?ET-1,NO and NOS?decreased.It can be observed in medicated group that,compared with those of the model group,serum E₂ decreased and P increased,and ovarian IL-6,TNF-2a,ET-1 and ET-1/NO decreased,while NO and NOS increased.Partial correlation analysis showed that serum E₂ was positively correlated with ovarian IL-6 and ET-1,and P was negatively correlated with IL-6.It is suggested that the disordered levels of reproductive hormones in the model rats of blood stasis due to cold syndrome might be related to the increase of the level of IL-6 and the activity of ET-I,and Shaofu Zhuyu decoction may improve the ovarian endocrine function by adjusting the levels of ET-1 and IL-6.?3?Effects on the oxidative damage to uterusThe results showed that,compared with those of the control group,uterus SOD and PGE₂ of the model group decreased,while COX-?,PGF_2? and MDA increased.It can be observed in medicated group that,compared with those of the model group,uterus SOD and PGE₂ increased,while COX-?,PGF_2? and MDA decreased;the differences were significant.Furthermore,SOD was positively correlated with PGE₂,and negatively correlated with COX-?and PGF2a;MDA was negatively correlated with PGE₂,and positively correlated with COX-?and PGF_2?.It is suggested that the oxidative damage of endometria should be closely related to the disabled smooth muscle contraction function.Shaofu Zhuyu decoction could adjust the levels of causing pain factors?PGE₂,PGF2a and COX-?,etc.?and the activities of oxidative stress factors,promote the anti-oxidation ability of endometria,recover the oxidative damage to uterus from cold,improve the function of endometria and relieve the pains of dysmenorrhea.