Sodium Tanshinone ⅡA Sulfonate Attenuates Angiotensin Ⅱ Induced Cardiac Fibrosis In Rats Via Keap1-Nrf2 Signaling

Background and ObjectiveCardiac fibrosis is characterized by the excessive accumulation of extracellular matrix proteins(ECM)in the cardiac interstitium,and can lead to both systolic and diastolic dysfunction.A growing body of evidence has shown that reactive oxygen species(ROS)and the renin-angiotensin-aldosterone system(RAAS)play a critical role in cardiac fibrosis.ROS can cause cardiac fibrosis through direct action on cells or indirect action via multiple signaling pathways.Angiotensin Ⅱ(Ang Ⅱ)is the major effector peptide of the RAAS and it can activate nicotinamide adenine dinucleotide phosphate oxidase(NADPH oxidase)which results in the production of ROS.The resultant imbalance between oxidant formation and elimination leads to oxidative stress.Increased oxidative stress can induce imbalances between ECM synthesis and degradation,along with collagen deposition in the cardiac interstitium,which leads to cardiac fibrosis.Upon the occurrence of oxidative stress eukaryotic cells develop an adaptive antioxidant stress response to sustain redox homeostasis.One of the key response factors is nuclear factor E2-related factor 2(Nrf2),which is a basic leucine zipper transcription factor that regulates various genes controlling the expression of proteins critical in detoxification and the elimination of ROS and electrophiles.It is widely accepted that Nrf2,when sequestered in the cytoplasm,is in its inactive state and is then associated with the ubiquitin E3 ligase adapter Kelch-like ECH-associated protein 1(Keap1)under normal conditions.This process leads to ubiquitination and degradation of Nrf2 and constitutively suppresses its activity.However,under oxidative stress conditions,Nrf2 dissociates from Keap1,translocates to the nucleus,and binds to the antioxidant response element(ARE).Subsequently,Nrf2 activates several downstream genes that encode enzymes with antioxidant action which increase the antioxidative ability of cells.Tanshinone ⅡA(Tan ⅡA)is one of the major active components extracted from Salviae miltiorrhizae(Danshen),which has been used in traditional Chinese medicine for many years.Owing to the high hydrophobicity of tanshinone ⅡA,sodium tanshinone ⅡA sulfonate(STS)was synthesized to increase its bioavailability.STS have been widely used in clinical practice due to its multiple cardiovascular therapeutic functions including anti-oxidative,anti-fibrosis,anti-atherosclerosis,anti-platelet aggregation and cardioprotective functions.However,the mechanisms underlying the anti-fibrosis and anti-oxidative effects of STS still remain unclear.In the present study,we inserted osmotic pumps containing Ang Ⅱ into the interscapular region to induce cardiac fibrosis.The rats were then treated with different doses of STS in order to investigate whether STS can attenuate Ang Ⅱ-induced cardiac fibrosis through activation of the Keap1-Nrf2 signaling pathway.Methods40 male Sprague-Dawley rats were randomly divided into a control group,Ang Ⅱ group,low-dose STS group,medium-dose STS group,and high-dose STS group.Osmotic pumps containing Ang Ⅱ were inserted into the interscapular region to induce cardiac fibrosis.STS groups were administered STS in different doses by intraperitoneal injection for three weeks.H&E staining and Masson’s trichrome staining were used to determine morphological changes and collagen content.The effects of STS on the expression of Keap1,Nrf2,Collagen Ⅰ,Collagen Ⅲ,antioxidant and phase Ⅱ enzymes were examined using reverse transcription-quantitative polymerase chain reaction(RT-qPCR)analysis and western blot analysis.Malondialdehyde content,total glutathione content and total superoxide dismutase activities in the cardiac tissues were measured using colorimetric method.ResultH&E staining showed that inflammatory cell infiltration,accompanied by myocardial necrosis and myofibrillar loss,was present to a greater degree in the Ang Ⅱ group than in the control group.The degree of myocardial lesion was decreased with increasing STS dose treatment in different STS treatment groups.Notably,no significant myocardial lesion was observed in the high-dose STS treatment group.Masson’s trichrome staining showed that Ang Ⅱ infusion markedly enhanced interstitial fibrosis accumulation in the Ang Ⅱ group,as indicated by increased collagen deposition.In contrast,only a small amount of blue fiber could be observed in the control group.Among the Ang Ⅱ-infusion groups,collagen deposition and CVFs decreased progressively with administration of increasing doses of STS.To further investigate the protective effects of STS against collagen deposition,we examined the mRNA expression of Collagen Ⅰ and Collagen Ⅲ using RT-qPCR analysis.The mRNA levels of Collagen I and Collagen Ⅲ significantly increased in the Ang Ⅱ group.However,the mRNA levels of Collagen Ⅰ and Collagen Ⅲ were decreased in the medium-dose and high-dose STS groups.High-dose STS administration significantly reduced Collagen Ⅰ and Collagen Ⅲ protein expression compared with expression levels in the Ang Ⅱ group and low-dose STS group(P<0.001,P<0.001 and P<0.05,P<0.05,respectively).In the tissue obtained from the Ang Ⅱ group,Keap1 and Nrf2 mRNA levels were slightly increased.However,Keap1 mRNA levels decreased following STS treatment.In contrast,Nrf2 mRNA levels increased further upon STS administration.Keap1 and nuclear Nrf2 protein expression were slightly elevated in the Ang Ⅱ group tissue compared with expression in the control group tissue(P<0.01,P<0.05).No significant difference was observed in the protein expression of cytoplasmic Nrf2 between the control goup and the Ang Ⅱ group.Compared with the Ang Ⅱ group,Keap1 and cytoplastic Nrf2 protein levels in the medium-dose STS group were slightly decreased(P<0.01,P<0.05),but nuclear Nrf2 protein levels were increased(P<0.01).However,Keap1 and cytoplasmic Nrf2 protein levels in the high-dose STS group tissue were significantly lower than the level recorded in the Ang Ⅱ group and low-dose STS group tissue(P<0.001,P<0.01 and P<0.01,P<0.01,respectively).In contrast,nuclear Nrf2 protain levels in the high-dose STS group were significantly higher than the level recorded in the Ang Ⅱ group and low-dose STS group(P<0.001,P<0.05).In the Ang Ⅱ group,HO-1 mRNA expression increased slightly compared with the mRNA expression in the control group(P<0.05),whereas no significant difference was observed in NQO1 and GCLC mRNA levels.The HO-1,NQO1 and GCLC mRNA expression in the high-dose STS group were increased compared with the Ang Ⅱ group and low-dose STS group.Compared with the control group,MDA levels were significantly higher and SOD activities were significantly lower in the Ang Ⅱ group(P<0.01,P<0.01).STS treatment reduced MDA levels but increased SOD activities in the low-,medium-,and high-dose STS groups.Compared with the Ang Ⅱ group,the medium-and high-dose STS groups exhibited higher levels of total GSH(P < 0.05,P < 0.01).Conclusions1.STS can attenuate Ang Ⅱ-induced cardiac fibrosis;2.STS can stimulate the nuclear translocation of Nrf2 and increase Nrf2 nuclear accumulation in a dose-dependent manner;3.STS can alleviate Ang Ⅱ-induced oxidative stress in the cardiac tissue and increase the antioxidative ability of myocardial cells.