The Effect And Mechanism Of Calcium Sensitive Receptor Mediated Macrophage Polarization On Proliferation And Remodeling Of Vascular Smooth Muscle Cells In Hypertensive Rats

Object:Hypertension is a widespread chronic disease whose development is facilitated by low-grade vascular inflammation induced by innate immune cells.As a member of the G protein-coupled receptor superfamily,the calcium-sensing receptor（Ca SR）regulates the development of hypertension through phospholipid lipoidase C（PLC）,inositol 1,4,5-trisphosphate（IP3）and intracellular calcium[(Ca²⁺)_i]signaling pathways regulate secretion of parathyroid hormone（PTH）,maintain calcium homeostasis,and participate in inflammatory responses.In our previous study,we found that inhibition of Ca SR activates NLRP3 inflammatory vesicles（nod-like receptor protein 3,NLRP3）,an important player in the innate immune response,promotes the secretion of inflammatory factors,and plays a significant role in the polarization of macrophages in spontaneously hypertensive rats（SHR）,thereby exacerbating vascular remodeling.Therefore,this experiment will further investigate the specific role and mechanism of Ca SR-mediated macrophage polarization and its effect on the proliferation and remodeling of vascular smooth muscle cells（VSMCs）through paracrine effects.Methods:8-week-old WKY and SHR were selected,and rats were anesthetized with chloral hydrate（0.3 ml/100 g）,and primary peritoneal macrophages were aseptically extracted immediately after alcohol immersion,and blood was sampled from the abdominal aorta,after which the thoracic aorta was aseptically extracted and tissue block apposition method was used to VSMCs were cultured in primary culture;after the cell density reached about 80%,cell passages were cultured by trypsin digestion,andα-smooth muscle actin（α-Actin）-related antigens were identified.They were grouped according to the experimental objectives as follows:（1）To investigate the role of Ca SR in regulating the polarization of peritoneal macrophages during hypertension:primary peritoneal macrophages were divided into four groups.WKY,SHR,SHR+R568（10μM）,SHR+NPS2143（10μM）.The supernatant of peritoneal macrophages was collected as conditioned culture medium for VSMCs intervention.（2）To investigate the effect of Ca SR-regulated NLRP3inflammasome-mediated peritoneal macrophage polarization on the proliferative remodeling of hypertensive VSMCs,peritoneal macrophages were divided into five groups according to the above method:WKY,SHR,SHR+R568,SHR+NPS2143,SHR+NPS2143+MCC950（10μM）;the supernatant of peritoneal macrophages was collected for the intervention of VSMCs.（3）Exploring the signaling pathway by which Ca SR regulates NLRP3 inflammasome-mediated peritoneal macrophage polarization affecting the proliferative remodeling of hypertensive VSMCs:WKY,SHR,SHR+R568,SHR+R568+U73122（PLC inhibitor）（10μM）;the same peritoneal macrophage supernatant was collected for intervention on VSMCs.（4）The M1 type marker inducible nitric oxide synthase（i NOS）,CD86,and M2 type marker arginase-1（arg-1）,CD206 were detected by western blotting and immunofluorescence in rat peritoneal macrophages.（5）The expression of Ca SR and CD86,CD206 and we use immunofluorescence to detect their co-localization.（6）Changes in intra-abdominal calcium ion([Ca²⁺]_i)concentration in macrophages were detected by Fluo-3/AM fluorescent probe.（7）The levels of IL-1βand IL-10 in the supernatant of peritoneal macrophages were detected by ELISA,and the levels of PTH in rat plasma were detected by ELISA.（8）The expression of Ca SR,NLRP3,caspase-1（cysteine aspartate-specific protease）,IL-1β,chemokine receptor CCR2（chemokine（C-C motif）receptor 2）in peritoneal macrophages was detected by western blotting.（9）The cell viability of VSMCs after12h,24h and 36h of peritoneal macrophages supernatant interventions were detected by CCK8 assay at 12h,24h and 36h,respectively.（10）The proteins of vascular proliferation,remodeling and apoptosis in VSMCs（α-SMA,OPN,Bax,Bcl-2,PCNA）were detected by western blotting.（11）The proliferation of VSMCs was detected using EDU assay.（12）Changes in cell migration ability were detected by Transwell.Results:（1）Macrophage polarization:compared with the WKY group,the expression of M1-type polarization markers CD86 and i NOS protein was increased in the SHR group（P<0.05）,CD86 fluorescence intensity was enhanced（P<0.05）,and inflammatory factor IL-1βcontent was increased（P<0.05）;the expression of M2-type polarization markers CD206 and Arg-1 protein was decreased（P<0.05）,the fluorescence intensity of CD206 was decreased（P<0.05）,and decreased secretion of anti-inflammatory factor IL-10（P<0.05）;compared with the SHR group,the expression of M1-type polarization markers CD86and i NOS protein was decreased in the SHR+R568 group of peritoneal macrophages（P<0.05）,CD86fluorescence intensity was decreased（P<0.05）,and inflammatory factor IL-1βcontent was decreased（P<0.05）;M2-type polarization marker CD206,Arg-1 protein expression increased（P<0.05）,fluorescence intensity of CD206 was enhanced（P<0.05）,and anti-inflammatory factor IL-10 secretion increased（P<0.05）;the opposite trend was observed for SHR+NPS2143（P<0.05）;NPS2143 combined with MCC950intervention attenuated this effect,as evidenced by increased expression of M2-type polarization markers CD206 and Arg-1 protein（P<0.05）and enhanced fluorescence intensity of CD206（P<0.05）,while M1-type polarization markers CD86 and i NOS protein expression were reduced（P<0.05）and fluorescence intensity of CD86 was diminished（P<0.05）;compared with SHR+R568,combined with U73122 intervention offset part of the protective effect of R568 group（P<0.05）.（2）Expression of Ca SR and co-localization with CD86and CD206:immunofluorescence results showed that the expression levels of Ca SR were consistent with the trend of CD206:compared with the WKY group,the expression of Ca SR and CD206 in peritoneal macrophages in the SHR group decreased（P<0.05）,while the fluorescence intensity of CD86 increased（P<0.05）;compared with the group of SHR,the fluorescence intensity of Ca SR and CD206 in the SHR+NPS2143 group further decreased（P<0.05）and increased CD86 expression（P<0.05）;SHR+R568 group showed the opposite trend（P<0.05）.（3）Intracellular calcium ions:compared with the WKY group,the concentration of[Ca²⁺]_i was decreased in the SHR group（P<0.05）,and further decreased after the intervention of NPS2143（P<0.05）,while compared with the SHR group,the concentration of[Ca²⁺]_i was increased after the intervention of R568（P<0.05）,the concentration of[Ca²⁺]_i was reduced in the combined SHR+R568+U73122 intervention group compared with R568 alone（P<0.05）.（4）CCR2 expression:western blotting results showed that the expression level of CCR2 protein was obviously higher in the SHR group compared with the WKY group（P<0.05）,and the expression of CCR2 protein was further increased in the SHR+NPS2143 group compared with the SHR group（P<0.05）,while SHR+R568 was able to inhibit this effect（P<0.05）.（5）Expression of NLRP3,caspase-1,and IL-1β:compared with the WKY group,NLRP3,caspase-1,and IL-1βprotein expression was significantly higher in the SHR group（P<0.05）;the expression of the above proteins was significantly lower in the SHR+R568 group compared with the SHR group（P<0.05）,and the SHR+NPS2143 group showed the opposite trend（P<0.05）;the SHR+NPS2143+MCC950group reversed the role of promoting NLRP3 inflammasome activation of NPS2143 compared with the SHR+NPS2143 group（P<0.05）;SHR+R568+U73122 attenuated some of the protective effects of R568compared with the SHR+R568 group（P<0.05）.（6）VSMCs proliferation:western blotting results showed that PCNA protein expression was significantly higher of VSMCs in the SHR group compared with the WKY group（P<0.05）and lower in the SHR+R568 group compared with the SHR group（P<0.05）;SHR+NPS2143exerted the opposite effect（P<0.05）.After administration of NPS2143 and MCC950 combined intervention,PCNA protein expression was reduced compared with the SHR+NPS2143 group（P<0.05）;PCNA expression was increased after R568 combined with U73122 treatment compared with the SHR+R568 group（P<0.05）.the EDU results were consistent with this（P<0.05）.（7）Apoptosis of VSMCs:the results of western blotting showed that compared with the group of WKY,the protein of the pro-apoptotic gene Bax protein was decreased（P<0.05）and the expression of the inhibitory gene Bcl-2 protein was increased（P<0.05）of VSMCs in the SHR group;after adding R568,the expression of the pro-apoptotic gene Bax protein was increased（P<0.05）and the expression of the inhibitory gene Bcl-2 was decreased（P<0.05）;SHR+NPS2143had the opposite effect to R568（P<0.05）;NPS2143 combined with MCC950 intervention increased Bax protein expression（P<0.05）and inhibited Bcl-2 protein expression（P<0.05）.Bax protein expression was decreased（P<0.05）and Bcl-2 protein expression was increased（P<0.05）when R568 was combined with U73122 intervention than R568 alone.（8）Phenotype conversion of VSMCs:compared with the WKY group,α-SMA protein expression was decreased（P<0.05）and OPN protein expression was increased（P<0.05）in the SHR group;compared with the SHR group,α-SMA protein expression was increased（P<0.05）and OPN protein expression was decreased（P<0.05）in the SHR+R568 group;the trend was reversed in the SHR+NPS2143.The SHR+NPS2143+MCC950 group showed increasedα-SMA protein expression（P<0.05）and decreased OPN protein expression（P<0.05）compared with the SHR+NPS2143 group,while the SHR+R568+U73122 group showed decreasedα-SMA protein expression（P<0.05）and increased OPN protein expression（P<0.05）compared with the SHR+R568 group.（9）VSMCs migration:Transwell results showed that the migration ability of VSMCs in SHR group was significantly higher than that in WKY group（P<0.05）,and the pro-migration ability was weakened after R568 intervention compared with SHR group（P<0.05）,while the opposite effect was observed in NPS2143（P<0.05）;the migratory ability of SHR+NPS2143+MCC950 group was decreased than the group of SHR+NPS2143（P<0.05）;SHR+R568+U73122 group（P<0.05）had an enhanced migratory ability compared to SHR+R568 group（P<0.05）.Conclusions:Ca SR activation promotes macrophage polarization to M2 and reduces polarization to M1,ameliorating hypertension-induced proliferation,phenotypic transition and migration of VSMCs,possibly by inhibiting NLRP3 inflammasome activation via the PLC signaling pathway.