Mechanism Of Kidney-tonifying Method Regulating KLF4 To Promote Endometrial Angiogenesis

The incidence of infertility is gradually increasing.In-vitro fertilization and embryo transfer(IVF-ET)technology is the main method to treat infertility,but the pregnancy rate of each embryo transfer is about 40%～ 60%,which is mainly due to low endometrial receptivity.So endometrial receptivity is important for the successful implantation of embryos.Angiogenesis is the key to improving endometrial receptivity.How to promote angiogenesis and improve endometrial receptivity has become one of the most important issues to be solved urgently in the field of reproductive medicine.KLF4,which belong to Krüppel-like factor(KLF)family,plays an important role in many pathophysiological processes such as cell proliferation,differentiation,tumorigenesis,vascular remodeling and inflammatory response.KLF4 is the key regulator of vascular homeostasis in endothelial cells and vascular smooth muscle cells.In different cellular contexts,KLF4 exhibits bidirectional effects of activation and inhibition in angiogenesis,which attracts much attention.In addition,as a transcription factor,KLF4 can interact with other proteins to regulate histone modification,and then activate or inhibit the transcription of target genes.Thus,KLF4 has an important role in angiogenesis.In the theory of traditional Chinese medicine,the kidney can store essence,regulate reproduction,and the essence can be transformed into blood,which directly provides the material basis for the development of the uterus and embryo.The endometrial receptivity is mainly regulated by the essence and blood of the kidney.Kidney-tonifying method is a common treatment method for infertility.This project intends to explore the effect of kidney-tonifying method on KLF4 and study the mechanism of kidney-tonifying method in regulating KLF4 to promote angiogenesis.This study has significant implications for illuminating the theory of “kidney dominating reproduction”.Part one Kidney-tonifying method promotes endometrial angiogenesis via KLF4Objective: To explore the effect of kidney-tonifying method on KLF4,and to study the effect of Kidney-tonifying method-induced KLF4 on the proliferation,migration and apoptosis of endometrial microvascular endothelial cells(HEMECs),which will clarify the mechanism of kidney-tonifying method promoting endometrial angiogenesis.Methods: The mice model of controlled ovarian hyperstimulation(COH)was established,and given with the kidney-tonifying method.The mice were divided into normal group,COH group and Bushen group.The uterus was harvested on the 4th day of pregnancy for HE staining to observe the endometrial morphology.Immunofluorescence staining was performed for the expression of α-SMA in uterus.The embryo implantation number of pregnant mice were observed on the 8th day of pregnancy.HEMECs were cultured and incubated with Bushen Tiaojing Decoction(BSTJD)serum and/or VEGFA,or infected with KLF4 overexpression or knockdown virus.Western blot and Quantitative Real-time PCR(q RT-PCR)were used to detect the protein and m RNA levels of PCNA,MMP-9 and Caspase 3.Scratch-wound assay were used to observe migration of HEMECs.The Ki67 expression was detected by immunofluorescence staining.HEMECs tubulogenesis was detected by tube formation assay.Results:1.Kidney-tonifying method improves endometrial receptivity and embryo implantation numberThe COH mice model was established.Compared with the normal group,the number of embryos implanted in model group was significantly reduced(P<0.05).Compared with model group,the number of embryos in the BSTJD group was significantly increased(P<0.05).HE staining showed that compared with the normal group,COH group had fewer uterine glands,sparse blood vessels,and dysplasia of the endometrium,while BSTJD treatment profoundly improved the endometrial morphology.Immunofluorescence staining showed that compared with the normal group,the number of α-SMA labeled vessels in the model group was significantly reduced.The number in BSTJD group was more than the model group.2.Kidney-tonifying method promotes angiogenesis in HEMECs.HEMECs were cultured and incubated with BSTJD serum.Western blot analysis showed that BSTJD could promote VEGFA protein expression.Tube formation assay showed that BSTJD could promote HEMECs tubulogenesis.3.Kidney-tonifying method promotes HEMECs the proliferation and migration of and inhibited its apoptosisWestern blot analysis showed that BSTJD increased the expression of PCNA and MMP-9 in HEMECs,and decreased the expression of Caspase 3.q RT-PCR were used to detect the m RNA levels of the above indicators,the results were the same as the Western blot.The expression of Ki67 was observed by immunofluorescence staining.The results showed that BSTJD could promote the expression of Ki67 in HEMECs.Scratch-wound assay showed that BSTJD could promote HEMECs migration.4.KLF4 mediates VEGFA-induced endometrial angiogenesis.Western blot analysis showed that VEGFA could promote the protein expression of KLF4 in a time-dependent manner.KLF4 overexpression was obtained by using Ad-KLF4 adenovirus infected with HEMECs,KLF4 overexpression further increased PCNA,MMP-9 expression by VEGFA,and decreased Caspase 3 in HEMECs.When HEMECs were infected with KLF4-specific sh RNAs(sh-KLF4)adenovirus to block endogenous KLF4 expression,VEGFA-induced expression of PCNA and MMP-9 and VEGFA-reduced caspase-3 expression was abrogated.Immunofluorescence staining showed that KLF4 could increase the expression of Ki67.Scratch-wound assay showed that KLF4 could promote HEMECs migration.These results indicated that KLF4 is essential for the induction of endometrial angiogenesis by regulating proliferation,migration and apoptosis responding to VEGFA signaling.5.Kidney-tonifying method promotes endometrial angiogenesis via KLF4Western blot and q RT-PCR analysis showed that BSTJD could increase the protein level and m RNA level of KLF4 in HEMECs.In order to study the role of KLF4 in promoting angiogenesis by kidney-tonifying method,HEMECs were infected with Ad-KLF4 virus to induce KLF4 overexpression,and given with VEGFA and/or BSTJD incubation.The results showed that BSTJD could promote the expression of PCNA and MMP-9,reduce the expression of Caspase3 in HEMECs.BSTJD promotes endometrial angiogenesis,improves endometrial receptivity,that lead to embryo implantation number improvement.BSTJD can coordinate with VEGFA to up-regulate KLF4 expression,which promotes HEMECs proliferation and migration,and inhibits cell apoptosis,that lead to promoting endometrial angiogenesis.Part two Kidney-tonifying method induced interaction between KLF4 and GCN5 to promote the expression of VEGFAObjective: Focus on KLF4 regulating VEGFA,to explore the epigenetic mechanism of BSTJD promoting VEGFA.Methods: The protein levels of VEGFA,KLF4,GCN5,succinylated H3K79 and the changes of MAPK signaling pathway in HEMECs were detected by Western blot.The interaction of GCN5 and KLF4 was detected by co-immunoprecipitation(Co IP).Double immunofluorescence staining was performed to observe the localization of KLF4 and GCN5.Chromatin immunoprecipitation(Ch IP)assay was used to detect the enrichment of KLF4,GCN5 and succinylated H3K79 on the VEGFA promoter.The succinylation of KLF4 was performed by immunoprecipitation(IP).The effects of GCN5 and KLF4 on VEGFA promoter activity were detected by luciferase assay.Results:1.Kidney-tonifying method promotes the expression of VEGFA in HEMECs by KLF4.HEMECs were infected with Ad-KLF4 and sh-KLF4 viruses,Western blot analysis showed that KLF4 overexpression increased the expression of VEGFA and KLF4 knockdown decreased VEGFA expression.When the cells with AdKLF4 were incubated with BSTJD and VEGFA,the protein level of VEGFA increased significantly.The results indicated that KLF4 was the key target of BSTJD to promote the expression of VEGFA.2.Kidney-tonifying method promotes the expression of VEGFA in HEMECs by GCN5Western blot analysis showed that VEGFA promoted the expression of GCN5 in a time-dependent manner.HEMECs were transfected with GCN5 overexpression plasmids(GFP-GCN5)and GCN5 knockdown plasmids(shGCN5).We found that GCN5 overexpression could increase the VEGFA protein level,and GCN5 knockdown could decrease VEGFA expression.The expression level of VEGFA was further increased when the cells incubated with GFP-GCN5 and BSTJD.BSTJD can synergistically promote VEGFA expression with GCN5.3.Kidney-tonifying method increases the interaction between KLF4 and GCN5To observe the effect of KLF4 and GCN5 on VEGFA promoter activity,luciferase reporter assay was performed.The results showed that KLF4 can activate the VEGFA promoter in a dose-dependent manner,while GCN5 cannot.The VEGFA promoter activity increased by KLF4 and GCN5 overexpression was higher than activity increased by KLF4 overexpression.Co IP revealed that the binding of KLF4 and GCN5 reached a peak when cells stimulated by VEGFA for 30 min.Immunofluorescence staining showed that KLF4 and GCN5 were almost all distributed in the cytoplasm before VEGFA treatment but translocated to the nuclear area in HEMECs treated with VEGFA for 30 min.Then we further observed the effect of BSTJD on the binding of KLF4 and GCN5,and found that BSTJD promoted the interaction between GCN5 and KLF4.At the same time,BSTJD promoted KLF4 and GCN5 from the cytoplasm to the nuclear area.Ch IP assay showed that BSTJD enhanced KLF4 enrichment in the VEGFA promoter region,while KLF4 overexpression increased the binding of GCN5 to the VEGFA promoter region.These results indicated that BSTJD could increase the interaction of KLF4 with GCN5,promote the KLF4-mediated recruitment of GCN5 to the VEGFA promoter,subsequently activating transcription which led to promote the VEGFA expression.4.Kidney-tonifying method promotes the succinylation of H3K79 by upregulating KLF4 and GCN5It has been reported that GCN5 can activate gene transcription by succinylating H3K79.To observe whether VEGFA could affect H3K79 succinylation,Western blot analysis showed that VEGFA time-dependently promoted H3K79 succinylation in HEMECs.When cells were transfected with GFP-GCN5,succinylation of H3K79 was enhanced.When cells were transfected with sh-GCN5,H3K79 succinylation was weakened,and VEGFA could no longer promote H3K79 succinylation.These results indicated that GCN5 could succinylate histone H3K79 in HEMECs stimulated by VEGFA.To observe whether KLF4 could regulate H3K79 succinylation,the cells were infected with Ad-KLF4 and sh-KLF4 respectively.Western blot analysis showed that KLF4 could promote H3K79 succinylation.Ch IP assay showed that GCN5 could enhance the enrichment of succinylated H3K79 at the VEGFA promoter.Then,we observed the effect of BSTJD on H3K79 succinylation,and found that BSTJD increased the level of succinylated H3K79 in HEMECs,and increased the binding of succinylated H3K79 to the VEGFA promoter region.These results suggested that BSTJD could promote the KLF4-mediated recruitment of GCN5 to the VEGFA promoter,subsequently succinylated H3K79 in the VEGFA promoter region.5.Kidney-tonifying method promotes the binding of KLF4 to GCN5 via the ERK signaling pathway.We further examined the effect of VEGFA on MAPK signaling in HEMECs.Western blot analysis showed that VEGFA could induce the rapid phosphorylation of P38,ERK and JNK in HEMECs.When HEMECs were preincubated with specific inhibitors of the above signaling pathways for 2h before exposure to VEGFA,the interaction between KLF4 and GCN5 were detected.The results showed that after administration of the ERK signaling pathway inhibitor PD98059,the binding of KLF4 with GCN5 were decreased.Then we incubated cells with BSTJD and VEGFA after PD98059 administration,and performed Co-IP assays.The results showed that the interaction between KLF4 and GCN5 in the VEGFA+BSTJD group was the highest among all groups,and the interaction between KLF4 and GCN5 in the VEGFA+BSTJD+PD98059group was lower than that in the VEGFA+BSTJD group,suggesting that BSTJD activated the ERK signaling pathway to promote the binding of KLF4 with GCN5.These results indicated that BSTJD activates the ERK signaling pathway to promote KLF4-GCN5 complex formation,which recruits GCN5 to the KLF4-binding sites of the VEGFA promoter in HEMECs to succinylate H3K79,transactivate VEGFA expression,leading to endometrial angiogenesis.Conclusions:1.Kidney-tonifying method promotes endometrial angiogenesis,improves endometrial receptivity,that lead to embryo implantation number improvement.2.Kidney-tonifying method can coordinate with VEGFA to up-regulate KLF4 expression,which promotes HEMECs proliferation and migration,and inhibits cell apoptosis,that lead to promoting endometrial angiogenesis3.Kidney-tonifying method activates the ERK signaling pathway to promote KLF4-GCN5 complex formation,which recruits GCN5 to the KLF4-binding sites of the VEGFA promoter in HEMECs to succinylate H3K79,transactivate VEGFA expression,leading to endometrial angiogenesis.