Experimental Study Of Crocin's Inhibitory Effect On Proliferation And Phenotypic Switch Of Vascular Smooth Muscle Cells

Objective:Proliferation and phenotypic switch of vascular smooth muscle cells?VSMCs?is one of the key initiating factors in the occurrence and development of atherosclerosis.Platelet-derived growth factor-BB?PDGF-BB?can induce a variety of biological effects and promote the proliferation and phenotypic switch of vascular smooth muscle cells.Crocin is one of the main functional components of saffron?Crocus Sativus L.?,a medicinal plant.Studies have shown that crocin can inhibit the formation of atherosclerotic plaques.However,the effect of crocin on the proliferation and phenotypic switch of vascular smooth muscle cells remains unclear.The aim of this study was to explore the effect of crocin on PDGF-BB-induced proliferation and phenotypic switch of vascular smooth muscle cells and its underlying mechanism.Methods:1.Research Object:VSMCs of SD rat thoracic aorta?Derived from primary culture of smooth muscle cells from rat thoracic aortic media?.2.Experimental Grouping and Drug Treatment:?1?.To determine whether crocin has cytotoxicity to VSMCs.VSMCs were divided into control,10?M crocin,50?M crocin and 100?M crocin groups.The proliferation rate of VSMCs was determined by CCK-8 after 24 hours and 48 hours of drug treatment.?2?.To determine the effect of crocin on PDGF-BB-induced VSMCs proliferation and phenotypic switch.VSMCs were divided into control,PDGF-BB?20ng/ml?,PDGF-BB?20ng/ml?+crocin?10?M?,PDGF-BB?20ng/ml?+crocin?50?M?and PDGF-BB?20ng/ml?+crocin?100?M?groups.After 24 hours medication,the proliferation rate of VSMCs was determined by CCK-8,the expression level of VSMCs contractile phenotype marker proteins:smooth muscle actin-???-SMA?,calponin and synthetic phenotype marker protein:osteopontin?OPN?was determined by Western Blot and Immunofluorescence Staining was used to observe the average immunofluorescence intensity of intracellular?-SMA and myofibrillar arrangement in VSMCs.?3?.To determine the effect of PDGF-BB on JAK/STAT3 pathway.VSMCs were divided into control,PDGF-BB?20ng/ml?,PDGF-BB?20ng/ml?+AG490?10?M?and AG490?10?M?groups?AG490:JAK inhibitor?.The expression of the JAK/STAT3 pathway protein p-STAT3?p-,phosphorylated?and total STAT3 were determined by Western Blot after 24hours medication.?4?.To determine the effect of PDGF-BB on ERK/KLF4 pathway.VSMCs were divided into control,PDGF-BB?20ng/ml?,PDGF-BB?20ng/ml?+U0126?50?M?and U0126?50?M?groups?U0126:ERK1/2 inhibitor?.The expression of ERK/KLF4 pathway proteins p-ERK1/2,total ERK1/2,and KLF4 were determined by Western Blot after 24 hours medication.?5?.To determine the effect of crocin on JAK/STAT3 pathway and ERK/KLF4 pathway.VSMCs were divided into control,PDGF-BB?20ng/ml?,PDGF-BB?20ng/ml?+crocin?10?M?,PDGF-BB?20ng/ml?+crocin?50?M?and PDGF-BB?20ng/ml?+crocin?100?M?groups.The expression of JAK/STAT3 pathway proteins p-JAK1,total JAK1,p-JAK2,total JAK2 and p-STAT3,total STAT3 and the expression of ERK/KLF4 pathway proteins p-ERK1/2,total ERK1/2and KLF4 were determined by Western Blot after 24 hours medication.?6?.To determine whether JAK/STAT3 pathway and ERK/KLF4 pathway mediate PDGF-BB-induced VSMCs proliferation and phenotypic switch.VSMCs were divided into control,PDGF-BB?20ng/ml?,PDGF-BB?20ng/ml?+crocin?100?M?,PDGF-BB?20ng/ml?+AG490?10?M?and PDGF-BB?20ng/ml?+U0126?50?M?groups.After 24 hours medication,CCK-8 was applied to determine the VSMCs proliferation rate and Western Blot was used to determine the expression of contractile phenotype marker proteins?-SMA,calponin and synthetic phenotype marker protein OPN.3.Sampling and Primary Culture of VSMCs:After thoracic dissection,the thoracic aorta of rats were clipped and harvested,the aortic tunica media were dissected under microscope and cut into tissue blocks?approx.1 mm²?,then attached to the wall of the culture flask and incubated in DMEM medium containing 10%FBS at 37?C in 5%CO₂ incubator until VSMCs crawled out of the tissue blocks,then changed culture medium,passaged,and the well-grown VSMCs of the 5th-9th generations were applied to subsequent experiments.4.Cell Viability Assay:After 24 h serum-free culture,VSMCs were seeded in 96-well plates with 5000 cells/well.Different drug interventions were added in line with the grouping conditions,and after 24 h incubation,10?l CCK-8 solution was added to each well and incubated for an additional 2 h at 37?C.The absorbance of VSMCs at 450 nm was measured by a microplate reader and the cell proliferation rate was calculated.5.Western Blot Analysis:After medication according to grouping conditions,VSMCs were lysed with RIPA lysate,total protein was extracted.BCA assay was used to determine protein concentration and a standard curve was drawn to calculate the loading amount and the loading volume.Protein samples were denatured,loaded on SDS-PAGE and transferred to PVDF membranes followed by blocked with skim milk at room temperature.After washing,the membranes were incubated with the primary antibodies overnight.Then,HRP-labeled goat anti-rabbit/anti-mouse secondary antibody was added and after incubating for 1 hour at room temperature,signals were visualized using an enhanced chemiluminescence kit and densitometry analysis of the immunoblots was carried out using Image J software.6.Immunofluorescence Staining:Following treatment of VSMCs with PDGF and crocin,cells were fixed in 4%paraformaldehyde and permeabilized in 0.1%Triton X-100 at room temperature.Subsequently,cells were blocked with 2%BSA followed by incubation with??SMA antibody overnight.Then,cells were incubated with goat anti-rabbit IgG H&L Alexa Fluor?488 secondary antibody at room temperature for another 1 h.Finally,nuclei were stained with DAPI.Images were taken under a fluorescence microscope.The?-SMA mean immunofluorescence intensity was analyzed by Image J software.7.Statistical Analysis:All experiments were repeated at least 3 times.All data are expressed as mean±SD.Statistical analyses were performed using one-way ANOVA with Tukey's post hoc test.The value of P<0.05 was statistically significant.Results:1.VSMCs primary culture:SD rat thoracic aortic media tissue blocks were placed in a culture flask containing DMEM medium.Three days later,VSMCs could be seen creeping out of the tissue blocks.On the sixth day,VSMCs grew to confluence and presented a typical"valley-peak"-like growth.The morphology of VSMCs is fusiform,rhomboid,spindle or polygonal.2.Crocin has no cytotoxicity to VSMCs:CCK-8 results revealed that compared with the control group,the proliferation rate of VSMCs was not significantly affected by crocin?P>0.05?,regardless of the intervention concentration and treatment time.3.Crocin inhibits PDGF-BB-induced proliferation of VSMCs:The results of CCK-8 manifested that the proliferation rate of VSMCs induced by PDGF-BB was significantly higher than that of the control group?P<0.05?,but after intervention with crocin,the proliferation rate of VSMCs was gradually inhibited compared with the PDGF-BB group?P<0.05?,and the degree of inhibition was positively correlated with the concentration of crocin.4.Crocin inhibits PDGF-BB-induced phenotypic switch of VSMCs:Western Blot showed that the expression of contractile proteins?-SMA and calponin was significantly decreased,while the expression of synthetic protein OPN was increased in VSMCs induced by PDGF-BB compared with the control group?P<0.05?.However,after intervention with crocin,the induction of PDGF-BB was gradually counteracted,the expression of?-SMA and calponin was re-enhanced,while the expression of OPN was inhibited?P<0.05?,and the degree of inhibition was positively correlated with the concentration of crocin.5.Crocin enhances intracellular?-SMA mean immunofluorescence intensity and improves PDGF-BB-induced myofibrillar arrangement disorder of VSMCs:The findings of immunofluorescence staining disclosed that PDGF-BB not only decreased the mean immunofluorescence intensity of intracellular?-SMA compared with the control group?P<0.05?,but also interfered with the arrangement of myofibrils in VSMCs,resulting in disordered myofibrillar arrangement and loss of regularity.However,after the addition of crocin,the mean immunofluorescence intensity of?-SMA was gradually increased?P<0.05?,and the degree of enhancement was positively correlated with the concentration of crocin.Additionally,the misalignment of myofibrils was gradually improved and the regularity was restored.6.PDGF-BB activates JAK/STAT3 and ERK/KLF4 signaling pathways:Western Blot unveiled that the expression of p-STAT3 was enhanced and the ratio of p-STAT3/total STAT3 was correspondingly increased in VSMCs induced by PDGF-BB?P<0.05?,when compared with the control group.But,the expression of p-STAT3 was inhibited and the ratio of p-STAT3/total STAT3 was also correspondingly decreased after AG490 intervention?P<0.05?.Also,compared with the control group,PDGF-BB enhanced the expression of p-ERK1/2 and KLF4 and increased the ratio of p-ERK1/2/total ERK1/2?P<0.05?,and both p-ERK1/2 and KLF4 expression were inhibited after U0126 was added for intervention,and the ratio of p-ERK1/2/total ERK1/2 was also decreased?P<0.05?.7.Crocin inhibits PDGF?BB?induced activation of JAK/STAT3 and ERK/KLF4 signaling pathways:Western Blot showed that the expression of p-JAK1,p-JAK2,and p-STAT3 in VSMCs induced by PDGF-BB was enhanced when compared with the control group,and the ratios of p-JAK1/total JAK1,p-JAK2/total JAK2,and p-STAT3/total STAT3 were correspondingly increased?P<0.05?.However,the expression of p-JAK1,p-JAK2,and p-STAT3 were inhibited after crocin intervention.The ratios of p-JAK1/total JAK1,p-JAK2/total JAK2,and p-STAT3/total STAT3 were also decreased?P<0.05?,and the degree of inhibition was positively correlated with the concentration of crocin.Likewise,PDGF-BB significantly increased the expression of p-ERK1/2 and KLF4 and the ratio of p-ERK1/2/total ERK1/2?P<0.05?,but after adding crocin,the expression of p-ERK1/2 and KLF4 was inhibited,and the ratio of p-ERK1/2/total ERK1/2 was correspondingly decreased?P<0.05?,also the degree of inhibition was positively correlated with crocin's concentration.8.JAK/STAT3 and ERK/KLF4 pathways mediate VSMCs proliferation and phenotypic switch:CCK-8discovered that when VSMCs treated with PDGF-BB,the proliferation rate increased?P<0.05?,but after blocking JAK/STAT3 pathway with AG490 and ERK/KLF4 pathway with U0126,the proliferative effect of PDGF-BB was counteracted,and the VSMCs proliferation rate was significantly decreased?P<0.05?.Furthermore,simultaneous blockage of both pathways by crocin also significantly reduced the VSMCs proliferation rate?P<0.05?.Western Blot showed that PDGF-BB decreased the expression of contractile proteins?-SMA and calponin and enhanced the expression of synthetic protein OPN?P<0.05?.However,the effects of PDGF-BB were counteracted after the JAK/STAT3 pathway and ERK/KLF4 pathway were blocked by AG490 and U0126respectively,the expression of?-SMA and calponin was re-enhanced,and the expression of OPN was inhibited?P<0.05?.Moreover,simultaneous blockage of both pathways with crocin also enhanced the expression of the contractile proteins?-SMA,calponin and decreased the expression of the synthetic protein OPN?P<0.05?,that is,counteracted the phenotypic switch induction of PDGF-BB.Conclusion:1.PDGF-BB can activate JAK/STAT3 and ERK/KLF4 pathway in VSMCs.2.The JAK/STAT3 pathway and ERK/KLF4 pathway mediate PDGF-BB-induced VSMCs proliferation and phenotypic switch.3.Crocin prevents proliferation and phenotypic switch of VSMCs by inhibiting JAK/STAT3 and ERK/KLF4 pathways.4.Crocin can enhance intracellular expression of?-SMA and restore the regularity of myofibrilar arrangement in VSMCs,which can improve the damaged systolic function of synthetic VSMCs.5.Crocin has no cytotoxicity to VSMCs.