| Stroke is the second most fatal disease in the world,and it is the leading cause of death and disability among adults in China.It has the characteristics of high incidence,high disability rate,high mortality rate and high recurrence rate.It is one of the diseases seriously harmful to human health,of which ischemic stroke accounts for about 70%.Its pathological mechanism is very complicated,including nerve inflammation,apoptosis,oxidative stress and so on.After ischemic stroke,the astrocytes in the brain are the first to be damaged,releasing a large number of inflammatory factors,thus further aggravating the brain injury.A large number of studies have shown that inhibiting the inflammatory response of astrocytes may be one of the important ways to treat stroke in the future.In recent years,a large number of in-depth studies have been done on the treatment of ischemia stroke,but there is still a lack of effective treatment.Traditional Chinese medicine has certain advantages in the treatment of stroke,and its pharmacological effects of multi-targets and multi-pathways have aroused widespread concern in the medical community.Traditional Chinese medicine believes that stroke is mainly due to the imbalance of yin and yang,qi and blood disorder and other symptoms,the treatment should be resuscitation aromatics,wake up and recovery of consciousness.Musk is one of the commonly used aromatic resuscitation medicine,which has pharmacological effects such as improving neurological impairment,inhibiting inflammatory reaction,reducing brain edema,specifically opening the blood-brain barrier and so on.Because musk is a national first-class protected animal,the clinical use of natural musk is limited.Artificial musk plays an important role as a substitute for natural musk,but at present,there are few studies on artificial musk in the treatment of stroke,and its theoretical mechanism needs to be further studied.Objective:The purpose of this study was to explore the protective effect of artificial musk on oxygen glucose deprivation/reoxygenation injury of astrocytes from anti-inflammatory injury,and to study its anti-inflammatory mechanism based on HMGB1/TLR4/NF-?B signal pathway,so as to provide experimental basis for artificial musk in the treatment of ischemic stroke.Methods:1.Through the Traditional Chinese medicine inheritance support system,the most frequently used aromatic resuscitation medicine in the Chinese Medicine Prescription Dictionary are selected,and the rules of awaking medicine use are discussed.2.Primary astrocytes were cultured in vitro,and the model of OGD/R injury was established.(1)The positive rate of astrocytes was identified by immunofluorescence staining of GFAP;(2)The cell viability,LDH release and ROS expression were determined by CCK8,LDH and ROS detection kits to screen the safe dose and effective dose of artificial musk.3.The expressions of TNF-?,IL-1?,IL-6m RNA and their corresponding proteins were determined by RT-PCR and ELISA methods to explore the antiinflammatory effect of artificial musk on astrocytes after OGD/R injury.4.To establish a method for detecting the transfection of HEK 293 T cells in vitro,and to detect the inhibitory effect of artificial musk on NF-?B signal pathway by dual luciferase reporter gene method.5.The expression of HMGB1,TLR4,TRAF6 proteins,the phosphorylation of IKK?,I?B?,NF-?Bp65 proteins and the expression of NF-?Bp65 protein in nucleus were determined by Western Blotting method.The nuclear translocation of NF-?Bp65 in astrocytes after OGD/R injury was observed by immunofluorescence method.To explore the antiinflammatory mechanism of artificial musk on astrocytes after OGD/R injury.Results:1.Musk is the most frequently used aromatic resuscitation medicine in the Chinese Medicine Prescription Dictionary.The commonly used herbs compatible with musk are dispelling-wind medicine,blood-activating and stasis-resolving medicine,aromatic resuscitation medicine,expectorant medicine,sedative medicine and so on.2.Compared with control group,0.001-10 ?g/m L artificial musk had no significant effect on astrocytes(P>0.05),100 ?g/m L artificial musk could significantly reduce the cell viability of astrocytes(P<0.001),0.001-1 ?g/m L artificial musk had no obvious effect on LDH release(P>0.05),10-100 ?g/m L artificial musk had an increase in LDH release(P<0.001).The safe dose range of artificial musk to astrocytes is 0.001-1 ?g/m L.3.Compared with control group,there was no significant change in cell viability and LDH release during OGD4 h/R22 h of astrocytes(P>0.05);the cell viability of OGD6 h/R18 h decreased to about 60%,and LDH release increased to 140%;the cell viability of OGD12 h/R24 h decreased to 46%,and LDH release increased to 180%;the cell viability of OGD12 h/R24 h decreased to 30%,and LDH release increased to 240%(P<0.001).Therefore,the injury model of OGD 6 h /R 18 h was established in this experiment.4.Compared with OGD/R group,0.01-1 ?g/m L artificial musk could increase the cell viability and decrease the release of LDH and ROS of astrocytes after OGD/R injury(P<0.05,P<0.01,P<0.001).Therefore,this experiment selected 0.01-1 ?g/m L as the effective dose for follow-up study.5.Compared with OGD/R group,0.01-1 ?g/m L artificial musk could reduce the relative expression and release of m RNA of TNF-?,IL-1? and IL-6 in different degrees(P<0.05,P<0.01,P<0.001).6.Compared with OGD/R group,artificial musk of 0.01-10 ?g/m L could significantly inhibit the activity of TNF-? induced NF-?B signal pathway(P<0.001).7.Compared with OGD/R group,0.01-1 ?g/m L artificial musk could reduce the expression of HMGB1,TLR4 and TRAF6 protein in astrocytes after OGD/R injury(P<0.05,P<0.01),and inhibit the phosphorylation of I???,I?B? and NF-?Bp65 protein(P<0.05,P<0.01,P<0.001);and it could inhibit the nuclear translocation of NF-?Bp65 induced by OGD/R and reduce the protein expression of NF-?Bp65 in the nucleus(P<0.05,P<0.01).Conclusion:Artificial musk inhibits the expression and release of inflammatory factors after OGD/R injury in astrocytes.Artificial musk can reduce the expression of HMGB1,TLR4 and TRAF6 proteins in astrocytes after OGD/R injury,inhibit the phosphorylation of I???,I?B?,NF-?Bp65 proteins and the nuclear transfer of NF-?Bp65 subunits,and exert its neuroprotective effect by regulating HMGB1/TLR4/NF-?B signal pathway. |
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