| Part Ⅰ Explore the potential targets and pathways of Siwu Tang in treating premature ovarian failure based on network pharmacologyObjective:To clarify the main compounds of SWT.To explore the potential targets and pathways of SWT in the treatment of POF through network pharmacology.Methods:(1)High performance liquid chromatography was used to analyze the active components in SWT.(2)The potential targets of SWT in the treatment of POF were predicted based on the related database of network pharmacology,and the enrichment analysis of GO,KEGG,DO were performed for the potential targets.Results:(1)Nine compounds in SWT were identified.(2)26 main compounds and its 124 predicted targets of SWT were screened,also the 2910 predicted targets of POF.80 common targets were obtained.(3)GO enrichment analysis showed that SWT had a role in the treatment of POF mainly by participating in the biological processes such as response to estradiol,response to hypoxia and angiogenesis.(4)KEGG enrichment analysis showed that SWT treated POF by regulating estrogen signaling pathway,ovarian steroidogenesis,HIF-1 signaling pathway,apoptosis and VEGF signaling pathway.(5)DO enrichment analysis showed that SWT may have potential therapeutic effects on Alzheimer’s disease,arteriosclerotic cardiovascular disease and bone related diseases.Conclusions:(1)SWT may regulate apoptosis in POF through IL6,TNF,BCL2,BAX,CASP3,CASP9,TP53 and other targets.(2)SWT may regulate angiogenesis in POF through HIF1A,VEGF A,HMOX1 and other targets.(3)SWT may regulate ovarian steroidogenesis and estrogen signaling pathway through ESR1,ESR2,AKR1C3,CYP1A1,CYP1B1,PIK3CG and play a role in treating POF related complications.Part Ⅱ Explore the mechanisms of Siwu Tang in facilitating ovarian function through improving ovarian angiogenesis in POF miceObjective:To evaluate the therapeutic improvement of SWT in treating POF mice,and reveal the specific mechanisms.Methods:(1)The untreared healthy mice were divided into the group of Control,Model,L-SWT,M-SWT,H-SWT and DHEA randomly.A mouse model of POF was established by a single intraperitoneal injection of Cy.(2)Ovarian tissue sections were prepared by hematoxylin-eosin staining for counting follicles.ELISA was used to detect levels of serum hormone and biochemical indexes.Tissue clearing in vitro was used for three-dimensional building of ovarian vascular.Immunohistochemistry was used for count of ovarian microvessels and localization of angiogenesis related factors.RT-qPCR was used to detect the mRNA levels of Sod1,Keap1,Nfe212,Homox1,Vegfa,Fgfb,Pdgfb,Angptl,Angpt2,Stat3 and Hifla.Western blot was used to detect the protein levels of Nrf2,HO-1,VEGF,bFGF,PDGFB,Ang1,Ang2,STAT3,p-STAT3 and HIF-1α.(3)Statistical analysis was carried out on pregnancy and birth status of mice in each group,including levels of serum hormone,the rates of pregnancy,average number of implantation sites,number of newborn mice and average weights of them.Results:(1)The mice in the Model group showed significantly disordered estrous cycle,and the body weight and ovarian index of them were significantly reduced.Treatment with SWT or DHEA could restore the regular estrous cycle,body weight and ovarian index of POF mice.(2)Mice in the Model group had decreased number of growing follicles and increased number of atresia follicles.The level of serum E2 was significantly decreased and the level of serum FSH was significantly increased in the Model group.Treatment with SWT or DHEA could increase the number of growing follicles,reduce the number of atresia follicles,restore the level of serum E2,and reduce abnormally elevated FSH level.(3)The mRNA and protein levels of Nrf2 and HO-1 in the ovary were significantly decreased in the Model group.Treatment with SWT or DHEA could increase the expression of Nrf2 and HO-1 in the ovary of POF mice.(4)In terms of angiogenesis,the distribution and extension of microvessels in the ovary were impaired and the number of them was significantly reduced in the Model group.Also,the STAT3/HIF-1α/VEGF signaling pathway was significantly inhibited,and the mRNA and protein levels of related angiogenic factors(VEGF,bFGF,PDGFB and Angl)were significantly decreased in the Model group.Treatment with SWT or DHEA can improve ovarian angiogenesis in POF mice by activating the STAT3/HIF-1α/VEGF signaling pathway to promote VEGF production,and increasing the expression of related angiogenic factors.(5)Compared with the Control group,the rates of pregnancy,the average number of implantation sites,the number of newborn mice and their average weights in the Model group were significantly reduced.Treatment with SWT could significantly improve the above situations.Conclusions:(1)SWT can improve follicular development,and have potential protective effect on pregnancy and embryo development in POF mice.(2)SWT protects the ovary from Cy-induced oxidative damage by activating Nrf2/HO-1 signaling pathway.(3)SWT improves ovarian angiogenesis in POF mice by activating the STAT3/HIF-1α/VEGF signaling pathway and increasing the production of related angiogenic factors.Part Ⅲ Study on the mechanisms of Siwu Tang in protecting HUVEC based on transcriptome sequencingObjective:To observe the effects of 4-OOH-CY and SWT on cell viability of HUVEC,and to reveal the protective mechanisms of SWT on HUVEC damaged by 4-OOH-CY.Methods:(1)HUVEC was pretreated with different concentrations of SWT for 24 h,and then 4-OOH-CY was used to establish chemotherapy-damaged HUVEC.The chemotherapy-damaged HUVEC were divided into the group of Model,M_LSWT and M_HSWT.Besides,Control group and C_LSWT group were also set up.(2)The cell viability of each group was detected by CCK8.(3)Transcriptome sequencing was used to quantitatively detect the whole transcriptome in each group,and different enrichment analysis were performed.(4)The key target genes were verified by RT-qPCR.Results:(1)The use of 4-OOH-CY alone resulted in a significant reduction in the number of viable cells in the Model group,while the pretreatment of SWT could improved the number of viable cells significantly.(2)There were 1806 differentially expressed genes in the comparison group between the Model group and the Control group,217 differentially expressed genes in the M_LSWT group and the Model group,and 481 differentially expressed genes in the M_HSWT group and the Model group.Besides,there were 59 differentially expressed genes in C_LSWT group and Control group.(3)Regulation of transcription(DNA-templated)and response to unfolded protein were the two key biological processes in GO enrichment.The main differentially expressed genes involved in regulation of transcription(DNA-templated)were ATF3,CELSR2,ZFHX4,ZFHX3,POU2F1,L3MBTL1,KMT2D,FOS,DDIT3,TEAD1,ZNF587,NFIX,NSD1,ZFP90,ZNF141,ZNF37A,ZNF425,ZNF483 and ZNF596.The main differentially expressed genes involved in the response to unfolded protein were CHAC1,DDIT3,DNAJB1,DNAJB4,HSPA1A,HSPA1B,HSPA1L and HSPA6.(4)MAPK signaling pathway and protein processing in endoplasmic reticulum were the two important results in KEGG enrichment.The main differentially expressed genes involved in MAPK signaling pathway were CACNG8,FOS,DDIT3,DUSP1,GADD45B,HSPA1A,HSPA1B,HSPA1L,HSPA6,PDGFRB and TAOK1.The main differentially expressed genes involved in protein processing in endoplasmic reticulum were DDIT3,HSPA1A,HSPA1B,HSPA1L,HSPA6 and DNAJB1.(5)RT-qPCR showed that compared with the Control group,mRNA levels of DDIT3,FOS,GADD45A,GADD45B,DNAJB1,HSPA1L and SOCS3 were significantly increased in the Model group,while mRNA level of BCL2 were significantly decreased.Compared with the Model group,mRNA levels of DDIT3,FOS,GADD45A,GADD45B,DNAJB1,HSPA1L and SOCS3 were significantly decreased in the M_LSWT group and M_HSWT group,while mRNA level of BCL2 were significantly increased.Conclusions:(1)The pretreatment of SWT could ameliorate the damage caused by 4OOH-CY to HUVEC.(2)SWT could inhibit 4-OOH-CYinduced apoptosis by decreasing the mRNA levels of DDIT3,FOS,GADD45A,GADD45B,DNAJB1,HSPA1L and SOCS3 in HUVEC.(3)SWT may regulate p38 MAPK and JNK signaling pathways and regulate endoplasmic reticulum stress to ameliorate 4-OOH-CY induced apoptosis. |
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