Tanshinone Inhibits Neuronal Cell Apoptosis And Inflammatory Response In Cerebral Infarction Rat Model

BackgroundIschemic stroke is a common disease in the elderly,it is due to cerebral artery blockage and other causes of the corresponding brain ischemia and hypoxia,which leads to neuronal damage and dysfunction of the nervous system.The higher fatality rate and disability rate have made it become one of the major diseases endangering public health.Although thrombectomy and drug therapy such as recombinant tissue plasminogen activator and thrombolytic drugs have been widely used in clinical treatment of cerebral infarction,The treatment effect is still limited,and often limited by the side effects of the therapeutic time window and more.Therefore,in order to improve the quality of life and relieve the family burden,it is urgent to find a more effective treatment strategy.Although the pathophysiological mechanism of cerebral infarction has not yet been fully understood,it has been shown that accelerated neuronal apoptosis is associated with cerebral infarction.Previous studies have demonstrated that downregulation of B cell lymphoma 2(Bcl-2)and upregulation of Bcl-2 related X protein(Bax)are involved in neuronal apoptosis and neuronal damage in rats with cerebral ischemia/reperfusion injury.The Bcl-2 gene family plays a key role in regulating apoptosis,including a variety of apoptosis related factors,such as Bcl-2 and Bcl-xL(antiapoptotic proteins),and Bax and Bak(pro apoptotic proteins).The levels of Bcl-2 and Bax are in equilibrium under normal conditions.Overexpression of Bax increases the number of Bax two polymers and promotes apoptosis,whereas overexpression of Bcl-2 can separate Bax two polymers and promote the formation of Bax and Bcl-2 complexes,thereby inhibiting apoptosis.Therefore,Bcl-2/Bax ratio plays an important role in regulating cell survival and death.In addition,the inflammatory response may also contribute to the occurrence of cerebral infarction.It is well known that inflammation plays a major role in regional cerebral ischemia.Regional cerebral ischemia/reperfusion injury can cause severe infarction tissue and induce inflammatory reaction because the tissue can not be repaired.The stimulation of the Secondary inflammation can lead to further damage in the infarcted area,resulting in the accumulation of neuronal cell damage.Some scholars have shown that anti-inflammatory factors may protect against the effects of middle cerebral artery occlusion(MCAO)in rats.In clinical practice,proinflammatory factors such as interleukin 6(IL-6)and tumor necrosis factor alpha(TNF-)have also been shown to be associated with clinical efficacy in patients with acute cerebral infarction.Therefore,drugs directed against apoptosis and inflammatory response should be effective strategies for the treatment of cerebral infarction.Tanshinone(TSN)is a kind of lipid soluble rosin type two terpene compound in Salvia miltiorrhiza Bunge.It is also called the total tanshinone.It is the main extract of the root of Salvia miltiorrhiza Bunge.It is divided into Tanshinone I,Tanshinone II A of 15 kinds of ingredients according to its different chemical structure.As the most important active component of the active ingredient,TSN and its structural analogues has a wide range of pharmacological effects and has attracted the attention of pharmaceutical scientists.In recent years,the study of its antioxidant,antibacterial,anti-inflammatory,especially the treatment of cardiovascular and cerebrovascular diseases has become more and more thorough.It was found that Tanshinone ?A was a correlation between dantone and lipid peroxidation,methylguanine transferase activity and apoptosis.In addition,Previous studies have shown that pretreatment with Salvia miltiorrhiza can inhibit the expression of vascular adhesion molecule-1(VCAM-1)and inhibit the activation of NF-kappa B induced by TNF-a in human aortic endothelial cells,what proved the anti-inflammatory properties of Salvia miltiorrhiza.It has been reported that the aqueous extract of Salvia miltiorrhizareduces the neutrophil adhesion rate by inhibiting the expression of intracellular-1 molecules by endothelin selectin,VCAM-1 and TNF-a.In addition,there are also reports that the enriched three terpene extract of Salvia miltiorrhiza can reduce the atherosclerotic lesion of aorta,which may be related to down regulation of CRP and monocyte chemoattractant protein.It is clear that TSN has been reported to inhibit levels of IL-12 and nuclear factor kappa B(NF-kB)in macrophages,In addition,TSN also inhibits the expression of TNF-a,IL-1? and IL-6 in LPS activated macrophages.All these results demonstrated the anti-inflammatory effects of TSN.Previous studies have confirmed that TSN can protect brain damage caused by ischemia/reperfusion.However,the mechanism of TSN on cerebral infarction has not yet been fully studied.ObjectiveThis research used the model of hypoxia in rats with cerebral infarction and glucose deprivation(OGD)neuron model to evaluate the effect of TSN pretreatment on cerebral infarction in rats with cerebral infarction volume,brain edema and nerve function defect,apoptosis and inflammation,so as to understand the TSN in vivo in vitro for cerebral infarction and the influence mechanism.Materials and Methods(1)A total of 45 healthy male Sprague-Dawley rats(250-280 g)were randomly divided into 3 groups(n = 15 for each group):normal control group(control group),middle cerebral artery occlusion and reperfusion(MCAO)group(MCAO group)and TSN pretreatment group(TSN group).Rats in the TSN group underwent intraperitoneal injections of TSN(5 mg/kg)daily for 7 consecutive days.After that,MCAO surgery was performed on the rats in the MCAO and TSN groups.Rats in the control group also underwent all surgical procedures for MCAO without ligation.(2)After 24 hours of reperfusion,the three groups of mice were evaluated by five grades and 4 points scoring methods.(3)We killed all rats after neurological evaluation and collected brain tissue samples from cerebral infarct areas in rats of the MCAO and TSN groups and from corresponding areas in rats of the sham group after 24 hours of reperfusion.And we measured the water contents of three groups of brain in order to evaluate the brain edema in each group.(4)Three groups of rats were killed and the brain was removed rapidly.And we sliced the forebrain along the coronal surface and use Image J analysis software to measure the volume of cerebral infarction in each slice of the two groups.(5)We collected their hippocampus and Cerebral cortex after killed the three groups of rat.After dewaxing and dehydration,TUNEL method was used to detect DNA fragments in the tissue system using the in situ cell death assay kit.And observed and recorded the apoptosis of three groups of samples with light microscope.(6)The levels of TNF-a,C-reactive protein(CRP)and IL-6 were measured by the corresponding enzyme-linked immunosorbent assay(ELISA)kits in three groups of rat cortical tissues.(7)Primary neurons were isolated from 5 newborn rats born about 1 day old.Neuronal cells were divided into control,hypoxia and glucose deprivation(OGD)and TSN groups.Cells in the TSN group were cultured in DMEM with 5 ?g/mL TSN for 5 days.Then,OGD was performed in the OGD and TSN groups.The cells in the control group were cultured at 5%C02.After 4 hours of culture,the medium was changed with the original conditions.(8)The treated cells were cultured for 24,48,and 72 hours,respectively.Each day,MTT(10 L,5 mg/mL)reagents were added to each culture dish,and the cells were cultured for 4 hours.The cell viability was detected by automatic quantitative mapping microplate reader.Annexin V-FITC apoptosis kit was used to evaluate the apoptosis.(9)The cells in the 3 groups were collected,and the levels of Bcl-2 and Bax were detected by Western blot and qRT-PCR.(10)SPSS 19.0 statistical analysis software(SPSS Inc.,Chicago,IL,USA)was used to analyze data.Continuous variables were expressed as the mean±standard deviation(SD)and analyzed by t test and one-way analysis of variance(ANOVA).A value of P<0.05 was considered statistically significant.Results(1)The results showed that compared with the sham group,brain water content,cerebral infarct volume,and neurological deficits scores were significantly increased in the MCAO group(all P<0.01).Pretreatment with TSN remarkably reduced brain water content(P<0.05),cerebral infarct volume(P<0.01),and neurological deficits scores(P<0.05)than those in the MCAO group.(2)The results revealed that MCAO operation significantly promoted neuronal cell apoptosis in hip-pocampus and cortical tissues compared with the sham group(all P<0.0 1),while cell apoptosis was significantly lower in the TSN group than in the MCAO group in hippocampus and cortical tissues(P<0.05 or P<0.01).(3)MTT results found that after 24,48,or 72 h of treatment,cell viability was significantly lower in the OGD group than in the control group(P<0.05 or P<0.01),while pretreatment with TSN obviously improved cell viability compared with the OGD group(P<0.05 or P<0.01).Flow cytometry results showed that compared with the control group,cell apoptotic rates were significantly increased in the OGD group(P<0.01),while TSN remarkably inhibited in the TSN group in comparison with the OGD group(P<0.01).In addition,both the mRNA and protein levels of anti-apoptotic protein Bcl-2 were significantly inhibited and pro-apoptotic protein Bax was obviously increased in the OGD group compared with the control group(P<0.01),while TSN remarkably reversed these changes in the expressions of Bcl-2 and Bax compared with the OGD group(P<0.05 or P<0.01).(4)ELISA results revealed that TNF-a,CRP,and IL-6 levels were all higher in the MCAO group than in the sham group(P<0.01),while reduced levels of TNF-a,CRP,and IL-6 were found in the TSN group than in the MCAO group(P<0.05 or P<0.01).ConclusionsIn this study,TSN was proved to protect against rat cerebral infarction,including cerebral infarct volume,cerebral edema,and neurological deficits.The apoptosis in hippocampus and cerebral cortex of rats with cerebral infarction,and the levels of IL-6,TNF-a and CRP were effectively inhibited by TSN pretreatment.In addition,TSN can significantly increase cell activity and inhibit apoptosis by downregulation of Bax and upregulation of Bcl-2 in OGD neurons.SignificationTSN has been widely used in clinical,in the treatment of infectious diseases,cardiovascular disease,tumor disease,diabetes,respiratory disease,facial features,oral cavity disease,bone disease,and disease of department of gynaecology,etc.,which have good curative effect.TSN preparation is convenient,safe and economical.It is absorbed by the intestinal tract and can be quickly distributed to the whole body.It can be used widely,with strong effect,slow excretion,no resistance and side effects.Previous clinical studies have also shown that TSN can treat cerebral infarction and has a unique therapeutic effect,but its mechanism has not been fully studied.This research has proved that TSN is by increasing the activity of neurons inhibit neuronal apoptosis and significant anti-inflammatory effects so as to achieve the mechanism of prevention and treatment of cerebral infarction,this provides a theoretical support for clinical drug use,and can indicate the potential of the treatment of cerebral infarction for the future research direction.DeficiencyBecause of various objective reasons,the research is still lack of largesample,multicenter,double-blind controlled large data such as support,And it is limited to animalexperiments or in vitro cell experiments;In addition,this reserch only studied the anti-inflammatory and anti-apoptotic effects of TSN.Different active ingredients such as Tanshinone I,Tanshinone II A and implicit Tanshinone's role of characteristics and effect mechanism still needs further research.These aspects will be the focus of my future research.