Mechanism Of Dihydroartemisinin Inhibiting Vascular Smooth Muscle Cell Proliferation And Inflammation

Dihydroartemisinin(DHA)has been globally recognized for its efficacy and safety in the clinical treatment of malaria for decades.Dihydroartemisinin(DHA),a semi-synthetic derivative of artemisinin,is a more water-soluble and effective anti-malarial agent than artemisinin.In addition to anti-malaria,DHA has also received increasing attention in other fields.DHA inhibits the proliferation,migration,inflammation,and angiogenesis of cultured endothelial cells.The anti-proliferative effects of DHA may be related to its interactions with vascular endothelial growth factor(VEGF)signaling.DHA induces sharp decreases in VEGF expression in several human cancer cell lines.In addition,DHA targets VEGFR2 in endothelial cells by regulating the NF-κB pathway,which inhibits angiogenesis.However,little is known about the underlying protective effects of DHA in Ang II signaling and the cellular and molecular mechanisms that affect gene expression-mediated vascular complications.Vascular smooth muscle cells(VSMCs),major components of the arterial wall,are responsible for maintaining vascular tone in response to hemodynamic changes and humoral stimulation.Angiotensin II(Ang II)is the primary effector of the renin–angiotensin system,which plays an important role in regulating blood pressure in pathophysiological conditions.Ang II can induce inflammation and proliferation of VSMCs,where the inflammation accelerates VSMC dysfunction.Ang II and other growth factors induce pathological conditions,including increased oxidative stress,inflammation,migration,hyperplasia,and hypertrophy,leading to VSMC dysfunction,which plays a critical role in the pathogenesis of hypertension,restenosis,and atherosclerosis.The fat mass and obesity-associated(FTO)protein belongs to the alk B family of α-ketoglutarate-dependent dioxygenases.As an obesity risk-associated gene and an m6 A eraser,FTO plays a critical role in promoting cell growth.Mice lacking FTO exhibit severe growth retardation.Additionally,mice deficient in FTO harbor specific bacterial signatures,which suppress inflammation.FTO plays an important regulatory role in RNA demethylation,which can regulate gene expression,DNA damage repair,and tumorigenesis.m6 A demethylation by FTO affects m RNA expression and stability.Zou et al.showed that the m6 A eraser function of FTO facilitates the proliferation and migration of human cervical cancer cells.In addition,m6 A modulation is involved in the proliferation and migration of VSMCs.However,the role of FTO in Ang II-induced inflammation and proliferation of VSMCs remains unclear.In this study,Ang II was used to construct VSMCs and vascular inflammation model in vitro and in vivo.The protective roles of DHA in inflammatory response and proliferation were evaluated through CCK-8,Brd U assay and immunofluorescence staining.The level of m RNA N6-methyladenosine was measured by m6A-RNA immunoprecipitation(Me RIP)assay.Western blot and quantitative real-time PCR were used to investigate the relationship between FTO and its potential downstream signaling molecules.Therefore,in this study,we aimed to determine the mechanism of Ang II-induced proliferation and inflammation of VSMCs and the associated protective role of DHA.Part One DHA inhibits Ang II-induced vascular smooth muscle cell proliferation and inflammatory responseObjective: To investigate the role of DHA in Ang II-induced vascular smooth muscle cell proliferation and inflammatory response.Methods:1.Various concentrations of DHA(0,1,10,20,and 100 μM)were added to VSMCs and cell viability was detected using the CCK-8 assay.2.Using CCK-8 assay and Brd U assay,the effect of co-adding VSMC with Ang II and different concentrations of DHA(0,1,10 and 20 μM)on their proliferation was examined.3.IL-6 and Ccl2 were selected as the factors to detect the inflammatory response.After adding DHA,q RT-PC was used to detect the expression levels of IL-6 and Ccl2 in VSMC and Ang II-induced VSMC,respectively.4.The Ang II infusion animal model was used to detect the expression level of IL-6 and the thickness of VSMC after adding Ang II or adding Ang II and DHA at the same time by double immunofluorescence method.Results:1.DHA inhibits Ang II-induced vascular smooth muscle cell proliferationDHA exhibited cytotoxic effects on VSMCs at 100 μM but not at lower concentrations.After VSMCs were stimulated with Ang II,cells were treated with different concentrations of DHA for 24 h,and cell viability was measured using CCK-8 assays.Ang II stimulation resulted in increased cell proliferation,whereas DHA attenuated the Ang II-induced cell proliferation in a dose-dependent manner.The anti-proliferative effects of DHA reached their maximum level at 20 μM.However,DHA treatment along cannot downregulate proliferation of VSMCs.Brd U staining confirmed that DHA suppressed Ang II-induced cell growth.2.DHA attenuated Ang II-induced inflammatory response in vascular smooth muscle cells.DHA-induced anti-inflammatory effects in VSMCs were further investigated using RT-q PCR.Ang II treatment significantly increased the m RNA levels of interleukin-6(IL-6)and C-C Motif Chemokine Ligand 2(Ccl2)in cultured VSMCs,whereas DHA decreased the m RNA levels of IL-6and Ccl2 induced by Ang II treatment,but not DHA treatment alone.3.DHA inhibits Ang II-induced inflammation in vascular smooth muscle cellswe performed an Ang II infusion animal model.Ang II infusion significantly elevated the IL-6 expression and increased the thickness of medial layer VSMCs.However,DHA treatment depressed the IL-6 expression and decreased the thickness of medial layer.These results indicate that DHA can significantly inhibit cell growth and inflammation in Ang II-induced VSMCs.Summary:1.DHA inhibits Ang II-induced vascular smooth muscle cell proliferation.2.DHA attenuated Ang II-induced inflammatory response in vascular smooth muscle cells.3.DHA inhibits Ang II-induced inflammation in vascular smooth muscle cell.Part Two FTO-mediated DHA inhibits vascular smooth muscle cell proliferation and inflammatory responseObjective: To explore the role and mechanism of FTO in the process of DHA inhibiting vascular smooth muscle cell proliferation and inflammatory response.Methods:1.Different concentrations of DHA were added to VSMC or VSMC induced by Ang II.The expression levels of FTO m RNA and protein were detected by RT-q PCR and Western blot.2.Two sh RNA vectors were constructed,and the two knockdown plasmids were transfected into VSMC cells,and the expression levels of FTO m RNA and protein were analyzed by RT-q PCR and Western blotting.3.VSMCs were transfected with sh FTO-2# or empty vector in the presence or absence of Ang II.The effect of FTO knockdown on VSMC proliferation was determined by Brd U.4.VSMC cells were treated with Ang II and DHA,and then double immunofluorescence staining was used to detect the expression levels of FTO and IL-6.5.Ang II-treated VSMC cells were transfected with sh FTO and blank vector,and the m RNA expression levels of IL-6 and Ccl2 were detected by RT-q PCR.6.The expression levels of KLF5 and p-GSK3β were detected by Western blot when DHA was added to VSMC or VSMC induced by Ang II.Results:1.DHA inhibits Ang II-promoted FTO expression in a dose-dependent mannerVSMCs were treated with different concentrations of DHA,in the presence or absence of Ang II.RT-q PCR were used to detect FTO m RNA expression.The result showed that Ang II significant elevated FTO m RNA level.However,this promotion can be depressed by DHA in a dose-dependent manner.In parallel,we found the same result in western blot analysis.2.Knockdown of FTO inhibits Ang II-induced vascular smooth muscle cell proliferationTransfection of cells with two different sh FTO plasmids decreased FTO m RNA and protein levels in VSMCs.Then,VSMCs were transfected with the sh FTO plasmid or empty vector in the presence or absence of Ang II.Brd U assays confirmed that depletion of FTO depressed Ang II-induced cell proliferation in VSMCs.3.Blocking FTO reduces Ang II-regulated vascular smooth muscle inflammationWe treated VSMCs with Ang II and DHA,and then double immunofluorescence staining detected the expression of FTO and IL-6.The result showed that DHA significantly reduced the expression of FTO and IL-6in VSMC.Ang II treatment increased the m RNA levels of IL-6 and Ccl2,and this effect was partially reversed by FTO depletion.Summary:1.DHA inhibits Ang II-promoted FTO expression in a dose-dependent manner2.Knockdown of FTO inhibits Ang II-induced vascular smooth muscle cell proliferation3.Blocking FTO reduces Ang II-regulated vascular smooth muscle inflammation Part Three DHA inhibits cell proliferation and inflammatory responses by modulating the FTO/NR4A3 axisObjective: To explore the mechanism through which DHA inhibits Ang II-induced cell proliferation and inflammatory response in VSMCs.Methods:1.VSMCs were treated with DHA in the presence or absence of Ang II,followed by RT-q PCR detection of m RNA expression levels encoding regulators that regulate VSMC cell proliferation and inflammatory responses.2.Cells were transfected with two different sh FTO plasmids,and the expression levels of NR4A3 m RNA and protein were detected by RT-q PCR and Western blot.3.VSMCs were transfected with wild-type and mutant FTO(H231A and D233A),and the expression levels of NR4A3 m RNA and protein were detected by RT-q PCR and Western blot.4.VSMC cells were transfected with sh FTO-1# and sh FTO-2#,and Western blot was used to detect the expression level of NR4A3 protein and the demethylation level of NR4A3 m RNA.5.VSMC cells were transfected with sh NR4A3-1# and sh NR4A3-2#,and the m RNA and protein expression levels of NR4A3 in VSMC were detected by RT-q PCR and Western blot.6.Ang II-induced VSMC cells were transfected with sh NR4A3-1# and sh NR4A3-2#,and the effects on their proliferation were detected by CCK-8assay and Brd U assay.7.sh NR4A3 or DHA alone,and both sh NR4A3 and DHA were added to VSMCs,and then RT-q PCR was used to measure the m RNA expression levels of IL-6 and Ccl2.Results:1.NR4A3 is a downstream target gene of FTOVSMCs were treated with DHA in the presence or absence of Ang II,and m RNA levels were measured.The result shown that NR1D1,NR4A3,and CCN1 were upregulated in Ang II-treated VSMCs.However,only NR4A3 was decreased after DHA treatment.This result implies that NR4A3 participates in FTO-regulated cell proliferation and inflammation in Ang II-treated VSMCs.Knockdown of FTO significantly suppressed the m RNA and protein expression of NR4A3 in VSMCs.To determine if FTO regulates NR4A3 expression through demethylation.2.FTO promotes the expression of NR4A3 by methylating m RNAWe transfected VSMCs with wild-type and mutant FTO(H231A and D233A).The result shown that overexpression of wild-type FTO,but not mutant FTO,increased the m RNA and protein levels of NR4A3 in VSMCs.These results indicate that the m RNA demethylase activity of FTO is critical for NR4A3 regulation.Furthermore,FTO knockdown increased the methylated level of NR4A3 m RNA,whereas FTO,but not mutated FTO,overexpression reduced the methylated level of NR4A3 m RNA.3.FTO/NR4A3 participates in DHA’s inhibition of Ang II-induced VSMC proliferationTransfection of VSMCs with sh NR4A3 significantly reduced the m RNA and protein levels of NR4A3 in VSMCs(Fig.5A and B).The depletion of NR4A3 also inhibited cell proliferation.Ang II-induced upregulation of NR4A3 protein was abrogated by knockdown of FTO in VSMCs.Furthermore,Brd U staining showed that DHA treatment decreased VSMC proliferation,whereas this effect was enhanced by concomitant depletion of NR4A3.4.FTO/NR4A3 axis mediates DHA to inhibit Ang II-induced VSMC inflammatory responseWe found that the expression of IL-6 and Ccl2 m RNA was decreased following NR4A3 knockdown or DHA treatment alone;the downregulation was significantly increased when NR4A3 knockdown occurred simultaneously with DHA treatment.Taken together,these results suggest that the FTO/NR4A3 axis is essential for the Ang II-mediated proliferation and inflammatory response in VSMCs and that DHA inhibits cell proliferation and inflammation by suppressing this axis.Summary:1.NR4A3 is a downstream target gene of FTO.2.FTO promotes the expression of NR4A3 by methylating m RNA.3.FTO/NR4A3 participates in DHA’s inhibition of Ang II-induced VSMC proliferation.4.FTO/NR4A3 axis mediates DHA to inhibit Ang II-induced VSMC inflammatory response.Conclusions:In this study,we found that DHA significantly inhibited Ang II-induced VSMCs proliferation and the expression of IL-6 and Ccl2 in a dose-dependent manner.Furthermore,we confirmed that fat mass and obesity-related(FTO)play a critical role in Ang II-induced VSMC proliferation and inflammation.FTO knockdown increased NR4A3 m RNA methylation levels,whereas non-mutated FTO overexpression decreased NR4A3 m RNA methylation levels.These results suggest that DHA plays a protective role in Ang II-induced VSMC proliferation and associated inflammation by inhibiting the FTO/NR4A3 axis.These data provide new insights into the mechanism of action of DHA and its critical role in the pathogenesis of hypertension-related vascular complications.