| Objective:To observe the effect of Tanshinone ⅡA in DOCA-salt hypertensive rats,to determine whether TanⅡA can attenuate DOCA-salt hypertension via suppressing oxidative stress and inflammatory response,so as to provide scientific basis for TanⅡA on the treatment of salt-sensitive hypertension.Methods:(1)Effect of Central administration of TNF-α on the expression of inflammatory mediators in the PVN of SD and Dahl-s rats.Adult male SD rats(250-280 g)and age as well as sex-matched Dahl-S rats were performed ICV microinjection with either TNF-α or saline,Three hours following ICV injection,rats were euthanized with overdose of isoflurane and then were used for either mRNA expression measurement or immunostaining,we test inflammatory mediators including IL-6,IL-1β、CCL5、CCL12、iNOS and p65.(2)Effect of Tanshinone ⅡA on primary neuron cells of SD and Dahl-s rats.SD or Dahl-S pup primary neuronal cultures were used to test the effect of TNF-αtreatment on inflammatory mediators’expression.Vehicle control or 2 ng/mL of TNF-αwere added to the culture medium and then cells were incubated in a 5%CO2 incubator for differing time periods(3,6,24 hours),at differing doses(0.2-20 ng/mL)of TNF-a.Real time PCR was then performed to test TNF-α induced inflammatory responses in the brain neurons,and then we test IL-6,IL-1β,CCL5,CCL12,iNOS,p65 mRNA level and immunostaining was also performed to test IL-1β,CCL5,iNOS and p-p65.Primary neuronal cultures were treated with different concentrations of TanⅡA(10μM,20μM,50μM)for 30 min,and then add 20ng/ml TNF-α,qPCR and immunostaning were performed to test above genes.(3)Effect of Tanshinone ⅡA sodium sulfonate in DOCA-salt hypertensive ratsThirty 8-week-old SD rats were randomly divided into 5 groups:control group,model group,Tanshinone ⅡA sulfonate low-dose group(10mg/kg.d),Tanshinone ⅡA sulfonate medium-dose group(15mg/kg.d),and Tanshinone ⅡA sulfonate high-dose group(25mg/kg.d).Except for the control group,other rats were subcutaneously implanted with DOCA pelets and given 1%NaCl+0.2%KCl water on the same day.Control group and model group were given intraperitoneal injection of equal volume of saline daily,and Tanshinone sodium ⅡA sulfonate were administered every day by intraperitoneal injection.Blood pressure was measured twice a week with non-invasive tail off.Weight BW once a week.On Day 22 rats were anesthesiaed,one of each group was perfused with PFA for HE staining,collected both kidneys and heart and then weighted;qPCR were performed to test IL-1β、IL-6、CCL5、CCL12、TNF-α、P65、Cyba、Cybb with both kidneys and left ventrical.Results:1.TNF-α central administration increases both SD rats and Dahl-s rats inflammatory mediators level in PVN,and the inflammatory response is augmented in Dahl-s ratsTNF-α central administration can increase CCL5,CCL12,IL-1β,IL-6,iNOS and p65 in both SD rats and Dahl-s rats.Compared with SD rats,the increase of CCL12 in Dahl-S rats was more significant(Dahl:15-fold SD:24-fold).Compared with SD rats,the increase of IL-1β was also higher,but not statistically significant(P=0.087),no significant difference was found in other genes.2.TNF-α treatment cause inflammatory response in both SD and Dahl primary neuron cellsTNF-α treatment significantly increased IL-1β,IL-6,CCL5,CCL12,iNOS and p65 mRNA level,the results of immunostaining is consistent with real time PCR,and inflammatory response to TNFα challenge is upregulated in the brain neurons of Dahl-S rats compared to SD rats3.TanⅡA can decrease inflammatory response and oxidative stress in primary neuron cultures.TanⅡA can decrease inflammatory response and oxidative stress.Different concentrations of TanⅡA(10μM,20μM,50μM)pretreatment can suppress p65 activation,decrease the mRNA level of proinflammatory cytokines(IL-1β,IL-6),chemokines(CCL5,CCL12),inducible nitric oxide synthase(iNOS)and NADPH oxidase subunit(Cyba,Cybb).The results of immunostaining is consistent with real time PCR.4.Tanshinone ⅡA sodium sulfonate can decrease the BP of DOCA-salt hypertensive rats,decrease the inflammatory response and oxidative stress of heart and kidney in DOCA-salt hypertensive rats.The BP of the model group was significantly increased,and the BP of TanⅡA sodium sulfonate group could be reduced to a certain extent,and the difference was statistically significant.The results confirmed that TanⅡA could decrease the blood pressure of DOCA-salt hypertensive rats.Compared with model group,the cardiac index and kidney index are decreased in treatment groups,it confirms that TanⅡA can reduce expansion of the left ventricle and kidney in DOCA-salt hypertensive rats,the mRNA level of IL-1β,IL-6 and TNF-α,CCL5,CCL12 are decreased in treatment groups,the mRNA level of P65 and Cyba,Cybb are also decreased in treatment groups.The results showed that inflammatory response and oxidative stress were increased in the model group,and the TanⅡA treatment group could partially inhibit inflammatory response and oxidative stress in DOCA-salt hypertensive rats.HE staining and in vitro photos of the kidney showed that the pathological changes of the kidney in the treatment group were also attenuated.Conclusion:1.Central administration of TNF-α could stimulate the inflammatory response in both SD and Dahl rats,Inflammatory Response to TNF-α Challenge is upregulated in the PVN of Dahl-S rats compared to SD rats,CCL12 is statistically greater in Dahl-S rats and the P value of IL-1β was 0.087,2.TanⅡA can inhibit the NF-κB(p65)activation,reduce the expression of PICs(IL-1β,IL-6),chemokines(CCL5,CCL12),inducible nitric oxide synthase(iNOS),therefore inhibiting the inflammatory response and oxidative stress in primary neuron cultures.3.TanⅡA sulfonic acid sodium can decrease the DOC A-salt hypertensive rats BP,inhibit the expression of NF-κB(p65),PICs(IL-1β,IL-6 and TNF-α),chemokines(CCL5,CCL12),inhibiting inflammatory response in DOCA hypertensive rats,and it also can decrease the oxidative stress(cyba,cybb),thereby protect DOCA-salt hypertensive rats. |
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