Long-term Protective Effect Of Nicotiflorin On Ischemic Stroke Animals

Ischemic stroke is a kind of cerebrovascular disease,which poses a great threat to human health.In recent years,the incidence of ischemic stroke in the world is gradually increasing,resulting in high mortality and disability rates.At present,interventional thrombolytic therapy is mainly used in clinical treatment of this disease.Although the effect is significant,this treatment method has relatively strict indications for patients,not all patients can be effectively treated,and most patients thus enter the long-term delay stage of stroke,with poor prognosis.At the same time,western drugs are mainly used in the clinical treatment of ischemic stroke,and these drugs are not satisfactory for the long-term prognosis of patients with ischemic stroke,and accompanied by a lot of adverse reactions.In last several years,the fact has proved that stroke,especially acute ischemic stroke,can be treated effectively by the traditional Chinese medicine,and the overall side effects are small.However,it is a pity that few studies have elaborated the protective mechanism of traditional Chinese medicine,and most of these studies focus on the short-term treatment of acute cerebral ischemia,while there is insufficient evidence for the long-term protective effect of traditional Chinese medicine on ischemic stroke.Therefore,the development of a drug that can provide long-term protection for patients with ischemic stroke is still an urgent and important topic to be solved in clinical application.Carthamus tinctorius L is a kind of traditional Chinese medicine which can typically promote blood circulation and make blood stasis be removed.It contains a variety of biological active substances,among which nicotiflorin,as a member of the flavonoid family in Carthamus tinctorius L,has attracted our attention.The research group has previously conducted in-depth studies on the role of nicotiflorin in acute cerebral infarction animal models,and proved that nicotiflorin can have an effective protective effect on cerebral ischemia-reperfusion injury rats in a short term.This paper mainly studied the long-term protective effect of nicotiflorin in the process of anti-ischemic stroke,to further confirm the long-term protective effect of nicotiflorin on stroke,and to provide scientific basis for the application of nicotiflorin in the prognosis of stroke.A model of cerebral ischemia reperfusion injury for 8 weeks is first established through middle cerebral artery occlusion(MCAO)in experimental animals,in order to simulate the clinical stage of long-term outcome after ischemic stroke.Then we observed the effects of nicotiflorin on survival rate and body weight change in rats during 8 weeks,the motion balance ability of experimental animals was tested by balance beam experiment and suspended tail deflection experiment,in the meantime,the effects of nicotiflorin on learning and memory ability of ischemic stroke rats were evaluated by water maze test.At the same time,we also observed the morphological function and structure of neurons in the hippocampal area of the rats at the 8th week by using Nissl staining and immunofluorescence staining.The results showed that nicotiflorin could improve the survival rate within 8 weeks of cerebral ischemia-reperfusion injury in rats,and the survival rate of rats increased by 33.3%after using nicotiflorin.In addition,there were no more deaths of rats in nicotiflorin group from day 9,while there were no more deaths of rats in model group until day 13.At the same time,the nicotiflorin group began to show a trend of weight gain in the first week after surgery,while the model group needed 2 weeks to recover the slow weight gain.That means the rats treated with nicotiflorin could make the weight grow faster than the model group,and beyond that,the value of weight at each observation time point was also significantly higher than the model group(P<0.05).In terms of neurobehavior,we observed that the contralateral deflection rate of rats in the nicotiflorin group gradually approached 50% over time,bilateral muscle strength tended to be symmetrical,behavior gradually coordinated,and motor ability was significantly improved.At the same time,the balance beam experiment shows that in the long-term prognosis stage,the nicotiflorin group got a more lower score compared with model group(P<0.01),the distance of slippage was less than 50%,and the body balance ability was significantly improved.In addition,the water maze test results showed that the incubation period of the rats injected with nicotiflorin reagent was shorter than that of the model group in the long-term prognosis stage after ischemic stroke,and in the statistics,the difference between those two groups was significant(P<0.05),meanwhile,the nicotiflorin group cross much more times through the hidden table position than that model group which had nothing to be treated(P<0.01),and what’s more,the result shows that no obvious difference was existed between the sham group and the group which was formed by rats that injected with nicotiflorin reagent.Simultaneously,the search trajectory diagram presented by the experimental results made us more intuitive that compared with the chaotic search mode of the model group,the nicotiflorin group rats could search for hidden platform through the combination of linear and trend search mode,which was closer to the normal group rats.These results suggest that nicotiflorin can improve the learning and memory ability of rats with neurological impairment caused by cerebral ischemia-reperfusion injury.In addition,through staining experiments,we found that nicotiflorin could also improve the morphological function of neurons in the hippocampal area of the brain of rats at week 8,keeping the structure of damaged neurons relatively intact and orderly.And also,nicotiflorin can increase the positive expression of microtubule-associated protein DCX and intermediate filament protein Nestin in neuron cytoplasm.The former protein is closely related to the migration and differentiation of nerve cells,while the latter is related to the regeneration of nerve cells.From there,it can go further promote nerve cell proliferation,differentiation and migration of the brain from ischemia-reperfusion injury after a long period of time in relatively good health condition.Secondly,in order to explore the effects of nicotiflorin on the gene level after cerebral ischemia-reperfusion injury in rats,we observed and analyzed the expression of related genes by transcriptome gene sequencing,and the results showed that compared with model group,1546 genes in nicotiflorin group were significantly different.These genes are closely involved in pathways such as inflammatory responses and programmed cell death.Among these genes,nicotiflorin was found to significantly down-regulate the expression of GSDMD,Caspase4,IL-18 and NLRP3.Among them,the expression level of GSDMD increased from2.105 to 13.530 after cerebral ischemia-reperfusion injury,and significantly decreased to3.460 after nicotiflorin application(P<0.01).The expression level of Caspase4 was decreased nearly 5 times from 11.560 to 2.370(P<0.01),and the expression level of IL-18 was also show a downward trend,which the specific value decreased from 13.250 to 2.865(P<0.05).These datas suggest that nicotiflorin may play an important role in regulating the above-mentioned inflammatory response and pyroptosis pathways,and also lays a foundation for further research.Thirdly,in order to further explore the long-term protective mechanism of nicotiflorin,we preliminarily studied the relationship between the brain protective effect of nicotiflorin and cell pyroptosis.For the purpose of figuring out the situation of expression that GSDMD,Caspase1,IL-1β and IL-18 have,which are key proteins relate with pyroptosis,we decided to use several experimental methods such as Western-blot and ELISA and so on.The results showed that nicotiflorin could significantly inhibit the expression of key pyroptosis protein GSDMD(P<0.01).However,it did not significantly regulate the expression levels of Caspase1,IL-1β and IL-18 proteins(P>0.05).Therefore,we preliminarily concluded that the mechanism of nicotiflorin playing its long-term protective role may be related to the inhibition of pyroptosis process,which can be interfered with by down-regulating GSDMD gene expression and inhibiting its expression at the protein level.However,the negative results of Caspase1 and others suggested that in the internal environment,the specific action mode of nicotiflorin in the process of pyroptosis is not known for us,and further experiments are needed to verify it.To sum up,the nicotiflorin has long-term protective effect on cerebral ischemia-reperfusion injury rats,it can improve the movement of ischemic cerebral apoplexy rats balance ability and the ability of learning and memory in a long time,make damaged nerve cells in the long-term prognosis of stage remains relatively intact state in order to maintain the corresponding neural function;At the same time,nicotiflorin can play a protective role by down-regulating the expression of the pyroptosis key gene GSDMD and inhibiting the expression of its protein level,thus delaying the process of pyroptosis.The results of this study provide a basis for nicotiflorin in the long-term protection of ischemic stroke.