| ObjectiveIn this study,streptozotocin(STZ)and arterial injury were used to prepare rat diabetes mellitus(DM)atherosclerosis(AS)model,and arterial injury rats were established as control group.To observe the body weight,blood glucose,arteriosclerosis degree,protein expression levels of toll-like receptor 4(TLR4),nuclear factor kappa B(NF-?B),nucleic acid-binding oligomerization domain-like receptor 3(NLRP3)inflammasome and downstream inflammatory factors in AS rats model with diabetes mellitus(DM).To explore the inflammatory mechanism of DM to accelerate AS,and compared with rosuvastatin,to elucidate the specific effectiveness and mechanism of ghrelin in the regulation of AS through the intervention of NLRP3 inflammasome and related inflammatory factors.MethodsA total of 45 SPF male Wistar rats aged four weeks,one week after adaptive feeding,the AS model was established by balloon injury to the left common carotid artery.In this process,five rats died,and 40 rats with successful modeling were randomly divided into four groups,10 in each group.Group A:carotid artery injury group;group B:diabetes group;group C:ghrelin group;group D:rosuvastatin group.Group A was fed with high-fat diet,and rats in groups B,C,and D were fed with high-fat and high-sugar diet,after 4 weeks,intraperitoneally injected with streptozotocin(STZ,40mg/kg)to prepare DM model,all DM model rats survived,after the DM model was successful,each group started intervention.Groups A and B were intraperitoneally injected with NS(100?g/kg,Qd),and group C was intraperitoneally injected with ghrelin(100?g/kg,Qd),group D rosuvastatin suspension gastric administration(5mg/kg,Qd),each group was administered once a day,and after 6 weeks,the rats were sacrificed.During this period,the blood glucose and body weight of rats were monitored regularly.HE staining was performed by carotid artery HE to observe the morphological changes of carotid artery.Western blot(WB)was used to detect NLRP3 and cysteinyl aspartate specific proteinase(caspase-1),TLR4,NF-?B,interleukin-1?(IL-1?)protein expression levels in rat carotid artery tissue,and correlation analysis of WB results of NLRP3,IL-1?,TLR4,NF-?B in ghrelin intervention group.Immunofluorescence was used to detect the expression levels of NLRP3 and TLR4 in carotid arteries.Results1.AS and DM modeling results(1)A total of 45 AS rats were prepared,of which 5 died.The cause of death may be excessive blood loss due to carotid vascular rupture during operation,improper injection of anesthetic in abdominal cavity,etc.The remaining 40 rats survived for 6 weeks and then sacrificed.HE staining of carotid arteries in all model rats showed different degrees of pathological manifestations of AS,suggesting that the AS model was successfully prepared.(2)A total of 30 rats with DM model were prepared.The blood glucose was measured on the 3rd and 7th day after STZ injection.The blood glucose of all model rats was greater than 16.7mmol/L,Model rats have no death,and there are different degrees of polydipsia,polyphagia,polyuria,rough hair,weight loss and other phenomena,consistent with the performance of diabetes,suggesting that the diabetes model was successfully prepared.2.Blood glucose and body weight results before and after intervention in each group of rats(1)Compared with the blood glucose before intervention,there was no significant change in blood glucose after intervention in group A,B and D(P>0.05),and blood glucose was significantly decreased in group C(P<0.05).(2)Compared with the weight before intervention,the weight of group A and C increased after intervention(P<0.01);the weight of group B significantly decreased after intervention(P<0.01);the weight of group D decreased after intervention,which was not statistically significant(P>0.05).3.Rats carotid HE staining resultsCompared with group A,the degree of carotid artery AS in group B was aggravated,the thickness of the carotid intima was significantly increased(P<0.01),the thickness of medial membrane was significantly decreased(P<0.01),and the ratio of intima-media was significantly increased(P<0.01);Compared with group B,the degree of carotid AS in group C and group D was alleviated,the thickness of the carotid intima was significantly increased(P<0.01),medial thickness significantly increased(P<0.01,P<0.05),and intima-media ratio significantly decreased(P<0.01,P<0.05);Compared with C,carotid artery AS was aggravated in group D,the thickness of the carotid intima was significantly increased(P<0.05),medial thickness significantly decreased(P<0.05),and intima-media ratio significantly increased(P<0.01).4.Expression of related proteins in carotid tissue of rats in each groupCompared with group A,the relative expression of NLRP3,caspase-1,IL-1?,TLR4 and NF-?B protein in group B was significantly increased(P<0.01);Compared with group B,the relative expression of NLRP3,caspase-1,IL-1?,TLR4 and NF-?B protein in group C significantly decreased(P<0.01);Compared with group B,the relative expression of NLRP3,caspase-1,TLR4 and NF-?B protein in group D significantly decreased(P<0.05),and the relative expression of IL-1? protein significantly decreased(P<0.01);Compared with group C,the relative expression of NLRP3 and IL-1? protein in group D significantly increased(P<0.01),and the relative expression of caspase-1,TLR4 and NF-?B protein significantly increased(P<0.05).5.Results of immunofluorescence of carotid artery in rats in group C and DThe expression of NLRP3 and TLR4 protein was observed in the intima and media of group C and group D,Compared with group C,the expression of NLRP3 and TLR4 protein in group D was significantly increased(P<0.01).6.Correlation analysis of NLRP3,IL-1?,TLR4 and NF-?B protein expression in group CCorrelation analysis was performed on the WB results of the above proteins in the ghrelin intervention group:NLRP3 was positively correlated with TLR4(r=0.7436,P=0.002),and NLRP3 was positively correlated with NF-KB(r=0.6874,P=0.001).IL-1?was positively correlated with TLR4(r=0.7763,P=0.008),and IL-1? was linearly positively correlated with NF-KB(r=0.8652,P=0.001).ConclusionDM is a risk factor for AS exacerbation or aggravation.In this process,NLRP3 inflammasome and related inflammatory factors play an important role.This study proposes new ideas for the mechanism study of AS.Exogenous ghrelin can inhibit the NLPR3 inflammasome to delay the progression of DM-induced AS.This effect may be related to the inhibition of the TLR4/NF-?B signaling pathway,and ghrelin may play a protective role in the progression of DM exacerbation or aggravation of AS by intervening in the above inflammatory pathways.Compared with the classic anti-inflammatory and anti-AS drug rosuvastatin,ghrelin has a more significant anti-inflammatory and anti-AS effect.In addition,ghrelin has a unique effect on the ability to maintain weight balance and lower blood sugar.This provides theoretical basis and data support for ghrelin in the clinical application of DM combined with AS. |
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