| Background and Objective:Myocardial fibrosis is a pathological manifestation of various cardiovascular diseases and a potential risk factor for cardiovascular events such as arrhythmia,heart failure and sudden cardiac death,but there is no specific medicine for myocardial fibrosis,so new compounds still need to be developed to prevent and treat cardiovascular diseases.At present,Chinese herbal medicine with multi-target effects have attracted wide attention due to lasting efficacy and small side effects,and Chinese herbal medicine also has anti-fibrosis effect,including cur cumin is a natural polyphenols and yellow extracted from spice turmeric.A large amount of evidence shows that cur cumin by affecting a variety of molecular targets to play anti-proliferation,anti-oxidation,anti-inflammatory and anticancer beneficial physiological effects,there have been reported that cur cumin can inhibit NLRP3 inflammation to achieve anti-inflammatory effect,and whether cur cumin can play an anti-fibrosis effect by inhibiting NLRP3 inflammation has not been studied.Therefore,this study constructs the myocardial fibrosis model,observed the effect of cur cumin on myocardial fibrosis and NLRP3 inflammatory body,and observed the role of NLRP3 inflammation in vitro model.Experimental method:Part One: 40 SD rats were randomly divided into four groups: control group,isoprenaline group,curcumin low-dose treatment group and curcumin high-dose treatment group.The rats in the control group were treated with pure water and normal saline injection,while those in the isoproterenol group were treated with pure water and isoproterenol injection.The low-dose curcumin group was treated with 100mg/kg curcumin for 4 weeks and treated with ISO injection in the last week,while the high-dose curcumin group was treated with200mg/kg curcumin for 4 weeks and treated with ISO injection in the last week.Take heart tissue as paraffin section,observe the pathological changes of myocardium by HE staining and Masson staining,and understand the degree of myocardial fibrosis;The relative protein expressions of Col1a1,α-SMA,NF-κB,NLRP3 and Caspase-1 in myocardium were measured by Western Blot.The relative expression of IL-1β m RNA in myocardium was detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction(q PCR).Part Two: the primary CFs of SD rats aged 1 ~ 3 days was extracted by differential adhesion method,and the cells were identified by immunofluorescence method,and then the 2 ~ 3 generations of cells were taken for experiment.The appropriate curcumin concentration was determined by MTT method.the cells were divided into control group,ang ⅱ group,angⅱ+curcumin low concentration group and ang ⅱ+curcumin high concentration group.the relative expression of Col1a1,α-SMA and protein in CFs was determined by Western Blot.The appropriate MCC950 concentration was determined by MTT method,and the cells were divided into control group,LPS+ATP group,LPS+ATP+MCC950 group,LPS+ATP+Cur group and LPS+ATP+MCC950+Cur group.the relative expression of α-SMA,NF-κB,NLRP3 and Caspase-1 protein in CFs and qrt-1 protein were detected by Western Blot.Experimental results:Part One:1.Masson trichromatic staining showed that myocardial cells in the control group were closely arranged and evenly distributed,interstitial collagen network was intact and collagen fiber content was low.In the model group,the arrangement of myocardial cells was disordered,a large number of myocardial cells were replaced by extracellular matrix,and myocardial fibrosis was obvious Compared with the model group,fibrosis after Cur treatment decreased significantly.2.HE staining results showed that the myocardial cells in the control group were normal in size,arranged neatly,stained uniformly in cytoplasm,and there was no inflammatory cell infiltration between myocardial tissues.In the model group,myocardial cells were hypertrophy and disordered,most myocardial tissues were swollen and loose,extracellular matrix increased,and a large number of inflammatory cells infiltrated.After Cur treatment,myocardial hypertrophy and infiltration of inflammatory cells in extracellular matrix were reduced.3.Compared with the control group,the protein expressions of Col1a1,α-SMA,NF-κB,NLRP3 and Caspase-1 in the model group were all up-regulated(P<0.05),and the above factors were all decreased after Cur treatment compared with the model group(P<0.05).4.Compared with the control group,the m RNA contents of IL-1β and IL-18 in the myocardial tissue of the model group were both up-regulated(P<0.05),and the m RNA contents of these factors were all decreased after Cur treatment(P<0.05).Part Two:1.Vimentin protein was identified as positive by immunofluorescence,which indicated that the extracted primary cells were cardiac fibroblasts.2.MTT results showed that the activity of curcumin decreased when the concentration was more than 50 u M(P<0.05),while the concentration of curcumin was 5-25 u M,which had no effect on the activity of cells(P> 0.05).Therefore,5u M and 25 u M were selected for the next experiment.However,MCC950 with the concentration of 10-1000 n M has no effect on cell activity(P>0.05),so according to the literature,50 n M concentration was selected for the next experiment.3.Compared with the control group,the protein expression of COLA1 andα-SMA in CFs was increased by Ang ⅱ stimulation(P<0.05),while Cur could inhibit the increase of COLA1 and α-SMA induced by Ang Ⅱ(P<0.05).4.Compared with the control group,the expression of α-SMA,NF-κB,NLRP3 and Caspase-1 protein in CFs was up-regulated in LPS+ATP group(P<0.05).Compared with LPS+ATP group,NF-κB and NLRP3 in LPS+ATP+MCC950 group had no obvious changes(P>0.05),but the expressions of α-SMA and Caspase-1 were down-regulated(P<0.05).The expressions of α-SMA,NF-κB,NLRP3 and Caspase-1 protein in LPS+ATP+Cur group were all down-regulated(P<0.05),while the expressions of α-SMA,NF-κB,NLRP3 and Caspase-1 protein in LPS+ATP+MCC950+Cur group were all down-regulated(P<0.05)Conclusion: 1.Curcumin can reduce myocardial collagen deposition and improve myocardial fibrosis in fibrosis rats.2.curcumin may improve myocardial fibrosis by inhibiting NLRP3 inflammasome activation. |
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