The Protective Effect And Mechanism Of Dan Hong Injection On Intracerebral Hemorrhage In Rat

Background and ObjectiveIntracerebral hemorrhage(ICH)is one of the most lethal stroke subtypes,The method of its therapy has not been elevated as well as that of ischemic.Rapid accumulation of blood within the brain parenchyma causes compression and primary brain damage.Then the interaction of cytotoxicity,excitotoxicity,oxidative stress(OS),and inflammation from the products of red blood cell lysis and plasma components caused secondary brain injury.Oxidative stress induces inflammation,while inflammation damage brain tissue through oxidative stress.Peroxiredoxins are a ubiquitous family of antioxidant enzymes that also control cytokine-induced peroxide levels which play dominant roles in regulating peroxide levels within cells.One kinetic measurement implys that Prxs reduce more than 90% of cellular peroxides.ICH stimulated Prx1 expression and extracellular release,TLR4/ NF-κB signaling activation,reflected by increases in TLR4 expression,extracellular signal-regulated kinase(ERK)1/2 and NF-κB activation,and production of cytokines.Dan Hong Injection(DHI)is derived from Chinese traditional medicinal materials Salvia miltiorrhiza and safflower.Its main active ingredients are hydroxy safflorin A,salvianolic acid B,and danshensu.Studies have shown that DHI can increase the levels of SOD and GSH in rats with cerebral ischemia-reperfusion injury,reduce the level of MDA,increase the expression of Nrf2,HO-1,and NQO1 against oxidative stress injury to protect brain tissue;DHI can promote traumatic cerebral hemorrhage.Hematoma absorption,improve the prognosis.Clinically,DHI is often used in ICH subacute phase(2 weeks later),which can promote hematoma absorption and improve the prognosis.However,most of the neurological deficits caused by ICH on individual is irreversible.Thus,we designed the experiment to verify the application of DHI in early stage of ICH through animal models,and explore the mechanism of anti-oxidation treatment of ICH,and provide a new strategy for the treatment of ICH.Methods Aged(18 months old)male Sprague-Dawley(SD)rats(weighing 600-700g)were randomly divided into(1)sham group,(1)vehicle-ICH group and(3)DHI-treated group.First,do a magnetic resonance image scan at 6h,24 h,48h,72 h,7d and 14 d after ICH model to obtain the lesion size over time.Second,1.0ml/kg sterilized water,sterilized water and DHI were injected to(1)(1)(3)group(12 rats in each)from 24 h,72h and 7d respectively(ip once daily,sustaining 3 days).Then record(1)mortality rate;(2)weight change;(3)neurological deficits(m NSS score):1d,3d,7d,14d;(4)brain MRI:1d,3d,7d,14d;(5)brain water content;(6)Cresyl Violet staining.Hematoma volume were compared between the three groups.The expression and its cellular location of Prx1 in the most effective group was verified by Immunofluorescence: Neu N,Prx1;GFAP,Prx1;and Iba-1,Prx1.Finally,Chelerythrine,as the MST1 agonist,was dissolved in dimethyl sulfoxide(DMSO)(10ul,1mmol/L).Again,rats were randomly divided into two group:(4)ICH+ Chelerythrine group(n=12);(5)ICH+DMSO(n=12).Chelerythrine was administered to the lesion core immediately after ICH,DMSO was administered as a vehicle control.DHI were given at 24 hours after ICH.Then look at the aforementioned index.Results1 The dynamic changes of MRI scanning,mortality,body weight and neurobehavioral deficits after ICHAt first,the MRI scanning at 6h,12 h,24h,48 h,72h,7d,14 d after ICH was obtained.The proportion of deaths was 14.29%,2/14 in the 14 th day after the model of rat brain hemorrhage.MRI showed that hematoma volume appeared differently with time after intracerebral hemorrhage in rats(Fig.1A-G).The change in body weight showed a gradual decrease in body weight from the third day after the model was established,an increase at the 7th day,and an improvement from the 7th day at 14 days(Fig.1H).Behavioural changes: The overall neurological function score showed a downward trend(Fig.1I).2 Observation on Curative Effect of Dan Hong Injection on aged Rats with intra Cerebral HemorrhageFrom the aspect of mortality,body weight,neurobehavior deficits,hematoma volume,brain water content after drug intervention.2.1 Effect of DHI on mortality,body weight and neurobehavioral deficits after ICHThere was no significant difference in the rat mortality rate(12.20%,5/41 vs 14.29%,6/42 vs 0%,0/36)after three groups of three time points intervention.Body weight: the DHI 1-3d group had significant changes at 7 and 14 days after ICH(Fig.2A,P<0.05);the DHI 3-5d group had a statistically significant increase in body weight at 14 days(Fig.2B,P<0.05),although there was improvement at 7 days,there was no statistical significance(Fig.2B,P>0.05);DHI 7-9d group had statistical significance at 14 days(Fig.2C,P<0.05).Behavioral changes: DHI 1-3d groups showed significant changes in 3 days after ICH(Fig.3E);m NSS decreased significantly in DHI 3-5d and DHI 7-9d groups on the 14 th day(Fig.3E-G,P<0.05).The changes of body weight and behavioral DHI 1-3d group were significantly different from those of DHI 3-5d group and DHI 7-9d group at 3d and 14d(Fig.2D/3H,P<0.05).2.2 Effect of DHI on hematoma volume,brain edema after ICHObserved by cranial MRI scan and CV staining,there were differences between the three time points after intervention(P < 0.05,Fig.4-5,Tab.2),but lesion size was significantly less than DHI at 14 days in the DHI 1-3d group.DHI 3-5d and DHI 7-9d group(Fig.5D/Tab.2,P < 0.05).Brain edema: Indirectly reflected by changes in brain water content,the DHI1-3d group was significantly smaller in the 14 th day after ICH than in the DHI 3-5d group,the DHI 7-9d group,and the vehicle group(Fig.6,P<0.05).3 Effect of DHI on oxidative stress via promoting Prx1 production after ICHIF showed the expression of Prx1 was mainly expressed in neurons in the first 3 days after ICH,and graually decreased with time after 12 hours of peaking,but was mainly expressed in astrocytes at 7 days and decreased at 14 days(Fig.7-10/11A).After administration of DHI,the expression of Prx1 in astrocytes was significantly increased,and its decline at 14 days was postponed(Fig.7/11 B,P < 0.05).4 Chelerythrine abolished the effects of DHI by phosphorylating and inactivating Prx1After administration of the inhibitor Chelerythrine,body weight gains in the 4th group were significantly smaller than those in the 5th group on the 3rd,7th,and 14 th days,m NSS score was not significantly lower than the control group,the lesion volume was not significantly reduced,brain water content was higher than that of the control group(Fig.12,P <0.05);brain MRI and CV staining showed that the volume of the hematoma in the 5th group was reduced,and no significant decrease was observed in the inhibitor group(Fig.13A/Tab.2,P<0.05).Prx1 expression was significantly reduced compared to the DHI group(Fig.14/ Fig.13 B,P<0.05).Conclusion1.The application of Dan Hong injection in the acute phase of cerebral hemorrhage in rats can promote the absorption of hematoma and improve the neurological function.2.Dan Hong injection exerts its anti-oxidation effect by promoting the expression of intracellular Prx1 in rat astrocytes.