| Ischemic stroke is one of the most common cerebrovascular diseases.With the aging of society,personal underlying diseases(such as hypertension,diabetes,heart disease,hyperhomocysteinemia),smoking,alcohol consumption and other factors,the incidence of IS has been continuously rising.At present,intravenous thrombolysis and mechanical thrombectomy are the main treatment methods for IS,however,rarely patients benefit from them for the strict time windows and equipments.The development of neuroprotective agents targeting the pathological mechanisms of IS is a therapeutic strategy for treating IS,but most of them have failed in their clinical transformation.Therefore,it is significant to explore deeper pathological mechanisms and develop new drugs for society and reality.The pathological mechanism of IS is very complex,including excitotoxicity,oxidative stress,inflammation,which are closely involved in the course of the IS.Among them,inflammatory response is considered the most important core axis.The IL-23/IL-17 axis is currently recognized as an important inflammatory response axis,widely involved in central nervous system(CNS)diseases including experimental autoimmune encephalomyelitis,traumatic brain injury,and IS.Previous research on the IL-23/IL-17 axis was mainly limited to the CNS,and researchers rarely focused on tissues or organs outside of the CNS.In recent years,the proposal of gut-brain axis has provided more possible pathological mechanisms for IS.Activation of the IL-23/IL-17 axis(including the expression of IL-23,IL-17 and IL-17+T cells)first occur in the intestine and then migrates to the CNS.This has shifted the focus on the IL-23/IL-17 axis to intestinal immunity.Traditional Chinese medicine is a huge treasure-trove and contains a large amount of unexplored therapeutic effects.Cordyceps sinensis is a traditional precious Chinese medicine with extensive pharmacological effects and broad prospects in the prevention of many major diseases.With clear anti-inflammatory and antioxidant effects,previous studies have found that Cordyceps sinensis has a significant protective effect on the IS mode.Cordyceps polysaccharide(CSP)is an important effective component of Cordyceps sinensis and can be extracted from artificially cultivated Cordyceps sinensis.Same as Cordyceps sinensis,it has confirmed that CSP have antioxidant and anti-inflammatory effects in modern pharmacology,and CSP should also have great potential in the intervention of IS.The objective of this project is innovatively to explore the intervention effect and mechanism of CSP on IS,providing new ideas for the deep development and research of traditional precious Chinese medicine.Objective:To explore the potential intervention of CSP on IS model rats and the in-depth mechanism.Method:1.Grouping SD rats according to a random number table(specific groups can be found in the materials and methods in each section).Using laser speckle to detect changes in blood flow before and after modeling.Detecting the volume of cerebral infarction in each group of rats using TTC staining.The modified neurological deficit scores(mNSS)were used to assess the neurological function of Middle cerebral artery occlusion(MCAO)rats.Observing the pathological changes of brain tissue in each group of rats by Hematoxylin and Eosin(HE)staining and Nissl staining.The brain edema of rats in each group were mearsured by calculating the cerebral water contents.Calculating the organ index using the ratio of organ mass to body weight.2.Observing the pathological changes of the ileum tissue by HE staining.Observing the changes in the distribution of glycoproteins in each group by Periodic Acid Schiff(PAS)staining.Western blot was used to detect the expression of ZO-1 in intestinal tissue.3.Western blot was used to detect the expression of TLR4,Myd88,P65,p-P65,IL-23,and IL-17 in intestinal tissue.Elisa kits were used to detect the expression of IL-6,IL-1β,TNF-α,IL-23,IL-17,malondialdehyde(MDA),Glutathione(GSH)and Superoxide dismutase(SOD).Result:1.The mNSS score showed that compared with the model group(MCAO group),the neurological deficit scores of the Naoxintong capsule group(NXTJN),Bailing capsule group(BNJN),Cordyceps sinensis group(CS),low dose CSP group(CSP1)、medium dose CSP group(CSP2)and high dose CSP group(CSP3)all showed improvements to varying degrees.The TTC results showed that compared with the MCAO group,all drug intervention groups showed reductions in cerebral infarction volume except for CSP1.Compared with MCAO group,the cerebral pathological morphology was improved,the number of Nissl bodies significantly increased,cerebral water content decreased and the brain organ index significantly decreased in each drug intervention groups.2.The results of inflammation and oxidative stress indicators in brain tissue of each group:Compared with Sham group,the MCAO group showed that IL-1β,IL-6,TNF-α,MDA significantly increased in ischemic brain of rats(P<0.01),while the GSH content and SOD activity decreased(P<0.01).Compared with MCAO group,CSP2 and CSP3 groups showed the expression of IL-1β significantly decreased(P<0.05).Compared with MCAO group,the expreesion of TNF-α and IL-6 significantly decreased(P<0.05).Compared with MCAO group,the GSH content in CSP2 and CSP3 groups was significantly increased(P<0.05,P<0.01),while the MDA content was significantly reduced(P<0.01).Compared with MCAO group,the SOD activity was significantly increased in CSP 1,CSP2,CSP3 groups(P<0.05).3.Compared with Sham group,MCAO group showed a decrease in spleen index(P<0.01)and an increase in liver index(P<0.01)in rats.Compared with MCAO group,all CSP groups significantly upregulated the spleen index(P<0.05,P<0.01,P<0.01).CSP1 and CSP3 can significantly lower the liver index(P<0.05,P<0.01).4.The results of intestinal HE and PAS staining showed that compared with Sham group,the MCAO group showed shed and necrotic epithelial cells,decreased glycoprotein secretion(P<0.01)and low-expressed ZO-1(P<0.01)in the ileum tissue of rats.Compared with the MCAO group,the pathological morphology of the ileum tissue in CSP2 and CSP3 groups of rats was significantly improved.And Compared with MCAO group,the glycoprotein secretion was increased(P<0.05,P<0.05)and the ZO-1 expression was up-regulated(P<0.05,P<0.01)in CSP2 and CSP3 groups.5.Western blot results showed that compared with MCAO group,the expression of TLR4,Myd88,IL-23,IL-17 and p-P65/P65 ratios in the ileum tissue of rats were significantly reduced in CSP2 and CSP3 groups(P<0.05 or P<0.01).6.The ELISA results showed that compared with the model group(MCAO group),the levels of IL-23 and IL-17 in ischemic brain of rats in CSP2 and CSP3 groups were significantly reduced(P<0.05 or P<0.01).Conclusion:1.CSP has obvious intervention effect on MCAO rats,which can improve the neuroethology score and pathological morphology in brain of MCAO rats and reduce the volume of cerebral infarction and brain edema.2.CSP can reduce inflammatory response and oxidative stress damage in ischemic brain.3.CSP protects the intestinal barrier and inhibits TLR4/Myd88/NF-κβ pathway and the expression of IL-23 and IL-17 in gut after IS.4.CSP inhibits the expression of IL-23 and IL-17 in ischemic brain. |
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