Study On The Protective Effect And Mechanism Of Refined Qingkailing And Its Components On Neurovascular Units In Vitro

Ischemic stroke is a major cause of death and long term disability worldwide.Ischemic stroke(IS)accounts for more than 80% of stroke.Previous study on in vitro usually target only a single cell type of the nervous system.With the continuous advancement of ideas and technology,we further emphasize the interrelationship between cells and cells.Since the concept of neurovascular unit(NVU)was proposed for IS in 2001,the roles,interactions,and dynamics of various cells and other components in the nervous system have been emphasized.Thus a variety of in vitro NVU models that mimic internal organs were constructed and studied under normal and pathological conditions,providing new ideas for finding new targets for clinical treatment.Qingkailing Injection(QKL),an excellent representative of traditional Chinese medicine injection,can effectively treat IS.Due to its complex and unclear composition,the difficulty of research on therapeutic mechanisms increase.We developed Refined Qingkailing injection(RQKL)consisting of geniposide(GEN),baicalin(BCL),cholic acid(CA)and hyodeoxycholic acid(HDCA).However,the protective effect and mechanism of RQKL and its components on in vitro NVU have not been revealed.Therefore,related researches were carried out in this research,with a view to providing new ideas for the treatment of IS based on in vitro NVU.Objective: The purpose of this study was to establish an in vitro NVU model consisting of high purity primary neurons,astrocytes and BMECs,as well as an in vitro blood-brain barrier(BBB)model consisting of high purity primary astrocytes and BMECs.Detecting the time of oxygen-glucose deprivation and re-oxygenation(OGD/R)injury on in vitro NVU model and in vitro BBB model oxygen to mimic IS in vivo.Studying the protective effects of QKL,RQKL,CA and HDCA on the in vitro NVU model after OGD/R and the protective effect of GEN on the in vitro BBB model after OGD/R.Observing the effect of BCL on the damage of OGD/Rinduced co-culture system through BDNF/TrkB/PI3K/Akt and BDNF/TrkB/MAPK/ERK signaling pathways.Methods: Neurons,astrocytes and BMECs were isolated from SD rats,and identified by immunofluorescence staining using specific markers of three types of cells(MAP2,GFAP and vWf),and then observed with microscope within 240 hours.The cell viability of different concentrations of astrocytes and BMECs after passage were detected by CCK8 method.Neurons were planted in the bottom of the culture plate at 0h.Astrocytes were planted on the outside of the membrane of the Transwell insert at 44 h.BMECs were planted into the inside of the membrane of the Transwell insert at 52 h.After 120 h(to 172 h),the function of the in vitro NVU model including the function of BBB and the state of neurons was examined to determine the success of in vitro NVU establishment.The function of BBB included fluorescent staining of tight junction protein(ZO-1),expression of tight junction proteins of ZO-1,occludin and Claudin-5(WB method),Transendothelial Electrical Resistance(TEER)values,permeability apparent(Papp)and Gamma-glutamyl transpeptidase(?-GT).The states of neurons included the phenotype under microscope and the expression of microtubule-associated protein 2(MAP2).To mimic IS conditions,the medium was replaced with deoxygenated Earle's balanced salt solution(EBSS)without glucose at 196 h.Then the NVU models were placed into the sealed Anaero container with oxygen indicator and an Anaero Pack for 1 hours to initiate the OGD insult.OGD was terminated at 197 h by complete medium and the cocultures were cultured under normal condition at 37°C for 24 hours to mimic reoxygenation.This process made an in vitro OGD/R injury model that mimicked in vivo ischemia-reperfusion.At 221 h,the function of in vitro NVU was tested to determine the success establishment of the injury model.Moreover,astrocytes were planted on the outside of the membrane of the Transwell insert at 0h.BMECs were planted into the inside of the membrane of the Transwell insert at 24 h to establish in vitro BBB.After 120 h(to 144h),the function of the BBB model in vitro was examined.After the in vitro BBB model was treated with OGD/R at 168 h,the function of in vitro BBB were examined.Different concentrations of QKL,RQKL,CA,HDCA,GEN and BCL were used to treat neurons,astrocytes and BMECs under normal culture conditions for 6h,12 h,24h and 48 h,respectively.The cell viability of the three cells was examined using the CCK8 method to determine the toxicity of cells.The above-mentioned drugs which were nontoxic were selected to interfere with different times of oxygen and glucose deprivation and reperfusion-treated individual cultured 3 types of cells.The cell viability was measured by the CCK8 method to determine the protection of the effect of drug.The established NVU models were randomly divided into 4 groups: Control group,Model group,RQKL high dose group(15.63 ?L/mL)and RQKL low dose group(0.98 ?L/mL).At 172 h,RQKL(15.63 ?L / mL and 0.98 ?L / mL)was used to pretreat in vitro NVU for 24 h,and then treated with OGD/R and the above concentration of RQKL,and divided into RQKL-H group and RQKL-L group.The in vitro NVU treated with OGD / R was taken as the Model group.The NVU Model treated only by OGD/R was the Model group.The NVU Model in the normal state without any treatment was the Control group.We examined the function of the in vitro NVU Model,as well as inflammatory factors(IL-1?,IL-6 and TNF-?),oxidative stress indicators(NO,MDA and SOD),lactate dehydrogenase(LDH),expression of apoptotic proteins(Bcl-2,BAX,caspase3 and caspase9)and neurotrophic factors(BDNF and GDNF).We respectively examined the protective effects of QKL(7.81?L/mL,2.54?L/mL),CA(93.75?g/mL,11.72?g/mL)and HDCA(10.16?g/mL,2.54?g/mL)on the NVU Model after OGD/R with the RQKL treatment method.The established BBB models were randomly divided into 4 groups: Control group,Model group,GEN high dose group(GEN-H group,25 ?g/mL)and GEN low dose group(GEN-L group,6.25 ?g/mL).At 144 h,GEN(25 ?g/mL and 6.25 ?g/mL)was used to pretreat in vitro BBB for 24 h,and then treated with OGD/R and the above concentration of GEN,and divided into GEN-H group and GEN-L group.The BBB models treated only by OGD/R was Model group.The BBB models in the normal state without any treatment was Control group.The BBB function and the apoptosis of BMECs in each group were detected,as well as the inflammatory factors,oxidative stress indicators,neurotrophin and LDH.The neurons were planted in the bottom of the culture plate at 0h.After 48 h,the astrocytes were inoculated into the inner side of the interpolated chamber.A co-culture system of neurons and astrocytes(NA)were established and co-cultured for 124 h.The NA models were randomly divided into 5 groups: Control group,Model group,BCL high dose group(BCL-H group,34.38 ?g/mL),BCL low dose group(BCL-L group,8.59 ?g/mL)and ANA inhibitor group.When the NA models were cultured for 172 h,incubation with BCL(34.38 ?g/mL and 8.59 ?g/mL)and ANA12(5 ?mol/L)for 24 h,followed by OGD/R treatment for 50 min,the NVU models of BCL intervention were BCL high dose group(BCL-H Group,34.38?g/mL)and BCL low dose group(BCL-L Group,8.59?g/mL).The NA modelS treated only by OGD/R was the Model group.The NA models in the normal state without any treatment was the Control group.The NA model that was treated with BCL(34.38?g/mL)and ANA12 was ANA-BCL group.The status of neurons in each group,as well as the inflammatory factors,oxidative stress and BDNF expression in the whole NA system were detected,and the expressions of TrkB,PI3 K,Akt,CREB,ERK1\2 and MAPK were further examined.Results : The purity of neurons,astrocytes and BMECs were >95,>99,and 100% respectively.The growth curves of three types of cells were obtained.An in vitro NVU model consisting of astrocytes,BMECs and neurons was successfully constructed by Transwell.The BBB barrier function in the in vitro NVU model was more integrity and neurons acquired a more mature morphological phenotype when cultured with astrocytes or BMECs than when cultured alone.OGD/R treatments were performed for different times on neurons,astrocytes and BMECs cultured separately.OGD1h/R24 h was determined to mimic the time of IS.We successfully constructed an in vitro BBB model consisting of astrocytes and BMECs using the Transwell platform.The BBB function of in vitro NVU model was more integrity.OGD1h/R24 h treatments was determined to mimic BBB injury after IS.The toxicity ranges of QKL,RQKL and the components of RQKL were obtained.The range of effective protective concentrations of QKL,RQKL and the components of RQKL were obtained,and two concentrations with significant differences in protection were chosen.QKL,RQKL and the components of RQKL maintains the integrity of the BBB after OGD/R by increasing the TEER value,the expression of tight junction protein and ?-GT activity,reducing the effect of Papp,and maintaining the neuron's state and MAP2.In addition,QKL,RQKL and the components of RQKL can also counteract inflammation,oxidative stress and apoptosis,as well as promote the release of neurotrophic factors.The protective effect of GEN on in vitro BBB was studied,which was found to increase TEER value,tight junction protein expression and ?-GT activity,and reduce the effect of Papp to maintain the integrity of BBB after OGD/R.It also acts against multiple mechanisms such as inflammation,oxidative stress and apoptosis,as well as the release of neurotrophic factors.Further studies have found that BCL can promote the expression of BDNF.After the specific inhibitor of BNDF,the ability of BCL against inflammation,oxidative stress and apoptosis is weakened.In addition,BCL is found to promote the expression of TrkB and CREB,the expression of MAPK and ERK1/2 in the downstream MAPK/ERK signaling pathway,and expression of PI3 K and Akt in the PI3K/Akt signaling pathway.Conclusion: QKL,RQKL and the components of RQKL acted against inflammation,oxidative stress and apoptosis,as well as the release of neurotrophic factors,as well as acted on multiple targets such as BBB and neurons in in vitro NVU.BCL can exert neuroprotective effects by regulating the BDNF/TrkB/PI3K/Akt and BDNF/TrkB/MAPK/ERK signaling pathways.