Changes In Cerebral Ischemia In Rat Blood-brain Barrier And P-glycoprotein And Qingkailing Effective Components Of The Role

For ischemic cerebral vascular disease no curative effect medicine can fundamentally solve the problem of injured brain. The damaged brain tissue impossibly gains the effective drug concentration because of the block of BBB. So the promotion of active drug transporting into the brain will become an important problem of waiting to be solved. In recent years, through the repeatedly clinical observation and the practice, it is thought that this disease belong to the category of'collateral disease'in Chinese medicine,'toxin hurts brain collaterals'is the key point in its pathogenesis, and remarkable clinical curative effect has obtained with the treatment targeting on the functional characteristic of collateral. Also, it is believed that the injury of brain vascular endothelial cell is the early pathological change and the basic agent of the attack of cerebral ischemia, and has close relationship with the development of cerebral vascular diseases. Modern medicine increasingly realizes that functional barrier composed by drug transporters plays an important role in the treatment of encephalopathy. Therefore, under the instruction of'collateral theory'of Chinese medicine and focus the target on effectively opening drug transporters in BBB for increasing the concentration of drugs , we further studied the relationship of P-glycoprotein in CMEC and the transport of active principles of Qingkailing and the interaction of them.Objective:Analyze and contrast active principles of Qingkailing in cerebrospinal fluid and serum from normal rats of different stages, and search the rule of active principles of Qingkailing going into and out of the brain tissue. Observe the morphologic change of BBB and function change of P-glycoprotein, and analyze medicine constituents influence the expression and function of P-glycoprotein. Then search the relationship of brain transport of active principles of Qingkaililng and morphologic and function change of BBB. This can reveal the compatibility effect of active principles of Qingkailing permeating ischemia BBB of injured brain in order to provide new thought for breaking through the bottleneck of developing drugs for the treatment of encephalopathy.Method:1. Observe the condition of permeation BBB of mainly active principles of Qingkailing: jasminoidin, baicalin, cholalic acid and hyodeoxycholic acid in Qingkailing injection are considered as analytic carrier of samples in vivo. We determine the changeable situation of active principles of Qingkailing in cerebrospinal fluid and serum of normal rats in different time points by the use of multi-fingerprint atlas.2. Observe the morphologic change of BBB in normal and ischemia rats of injured brain: HE slice and ultrathin slice of brain tissue are considered as investigation target, and with the two ways of light microscope(LM) and transmission electron microscope(TEM) analyze the change of morphosis of BBB and the situation of injured brain in normal rats and ischemia and reperfusion rats of different time points.3. Observe the differential characteristic of time series of the expression of P-glycoprotein in BBB of ischemic rats: with the way of western blotting and immunohistochemistry analyze the differential characteristic of time series of the expression of P-glycoprotein after cerebral ischemia and reperfusion of rats. And we made an ultramicrostructural position of P-glycoprotein by utilizing immunity electron microscope. Then in a certain degree this can reflect that the change of expression and function of the protein may cause the change of the permeability of BBB.4. The interaction of active principles of Qingkailing on P-glycoprotein in microvascular endothelial cells: firstly, we succeeded in cultivating and identifying RCMECs and observing profuse microvillus of endothelial cells by scanning electron microscope(SEM) and typical tight junction(TJ) by TEM. We also identifyâ…§factor by immunocytochemistry and the purity can reach 98%. Secondly, we take RBMECs in vitro culture as research object and analyze that active principles of Qingkailing influence the function and expression of P-glycoprotein in endothelial cells with MTT and western blotting.5. The influence of active principles of Qingkailing on the morphosis of BBB and expression of P-glycoprotein in the normal and ischemia and reperfusion rats: using TEM and immunohistochemistry we observed the ultramicrostructure of BBB and the expression condition of P-glycoprotein in vivo. The result revealed the compatible effects on penetrating to BBB by Qingkailing effective components from two points of view-structure and function.Result:1. Jasminoidin can be detected in two kinds of samples of body fluid-CSF and serum, which demonstrates Jasminoidin is able to penetrate BBB. At the same time Jasminoidin in two kinds of body fluid shows the identical tendency of accumulation and elimination, and the time of reaching the peak is from 0.2 to 0.5 hour. And that in CSF slightly delays, which is on 0.5 hour or so. From this point we can illustrate after drug in the blood reaches the peak time most of Jasminoidin is able to accumulate in CNS. But we can not determine baicalin, cholic acid and hyodeoxycholic acid in CSF, which may explain they can not reach through BBB. Because it is endogenous substance cholic acid and hyodeoxycholic acid forms two-humped phenomenon, which results from compensated cholic acid secreted in the body.2. By observing the morphosis of BBB in the state of ischemia and reperfusion we find that the time of reperfusion has a big relation with the permeability of BBB. At 0h and 0.5h after reperfusion the gaps around capillaries become existed. Hydropsia begins to appear and endothelial cells lightly swell. But TJ of BBB does not completely open. The organism goes to the stage of compensation and permeability increases slightly. At 2h and 6h after reperfusion the vacuolization in endothelial cells is obvious and there are inflammatory cells infiltrating, which demonstrates membrane permeability starts to increase. Meanwhile the distance between foot process of astrocyte and basal membrane has greatened. So the permeability of BBB further increases. At 12h and 24h after reperfusion because of the composed structure of BBB damaged greatly the permeability of BBB should reach the maximum degree at this time. At 48h and 72h after reperfusion most of endothelial cells are not integrated in shape, however, the gaps around capillaries become smaller and hydropsia lessens especially at 72h. Quantities of neurocytes and gliocytes have died and disintegrated and there are a large number of gitter cells in the brain tissue, which hints that reparation of the tissue has began and the permeability of BBB has degraded in some extent especially at 72h.3. The change of expression level of P-glycoprotein can be divided into three phases. In the first phase (from 0h to 6h) the expression level of P-glycoprotein reaches the lowest degree. The reason is that a lot of mediators of inflammation released cause the damage of endothelial cells at the early stage of reperfusion and the excreted function of BBB weakens due to decrease of the expression level of that. Although the expression level of protein obviously ascends at 0.5h owing to the compensation of the organism, it returns to the lowest point quickly. In the second phase (from 12h to 72h) the expression level of protein increases gradually and the augment of P-glycoprotein is faster than the recovery of BBB structure. The newly synthetical protein can assemble to the membrane quickly to compensate the raised permeability of BBB on account of disorganization. Then it can excrete many noxious substances for the maintenance of brain homeostasis. In the third phase (from 4d to 10d) the expression level of P-glycoprotein has obviously increased and approached to the normal because endothelial cells and astrocytes begin to repair due to dissociation and hyperplasia especially on the fourth day. The variance of expression level of P-glycoprotein may reflect the change of function in some extent.4. When the concentration of VCR is 10nM and 20nM jasminoidin and cattle cholic acid can oppose the cytotoxic action of VCR regardless of the normal cells or the handled cells. They are able to raise the cytoactive of endothelial cells. Synthetizing the effect of verapamil we can suppose that jasminoidin and taurocholic acid may make VCR pumped out of cells by enhancing the transport function of P-glycoprotein for protecting endothelial cells from the damage. However, when the concentration of VCR attains 30nM both of them can impossibly oppose this toxic action because this concentration is so high that the toxic action becomes more powerful. Having studied the contribution of action principles of Qingkailing to the expression of P-glycoprotein we discover that jasminoidin make no difference. However, to our surprised, taurocholic acid raises the action of cells by enhancing the expression level of this protein thereby excreting a lot of VCR. Baicalin and Qingkailing can not be against the effect of VCR and not protect endothelial cells. Moreover VCR+Q aggravate the cell damage in model group, which illustrate Qingkailing may cause determinate toxicity in this condition.5. In normal medicine group other action principles except baicalin have on effect on the structure of BBB contrast to normal control. We have observed three-layered structure is integrated and endothelial cells and foot process have no hydropsia and there are no obvious gaps around capillaries. Only in baicalin group we find perivascular spaces increase a little and the sign of hydropsia, which makes us consider baicalin may enhance the permeability of BBB in some extent. With regard to model medicine group three active principles can not improve the hydropsia obviously when used alone, but Qingkailing makes perivascular spaces small, makes hydropsia lightened and make the tissue damage lessened. So Qingkailing can lessen brain edema and lower the permeability of BBB contrast to other medicine groups. It can educe double-protective action.Conclusions:1. The time-change regulation of three active components of Qingkailing in serum including jasminoidin, baicalin, cholic acid and hyodeoxycholic acid is that the concentration gradually decreases along with time. Only jasminoidin can penetrate BBB, and the peak time in CSF delays slightly contrast to that in serum. So jasminoidin is the basic substance to awake mind and wake up a patient from unconsciousness and has an important role in the treatment of cerebrovascular disease. Therefore active principles of complex prescription educe the compatible effects on penetrating to BBB, and other drug components probably change the structure and function of BBB to open BBB specially. Then effective drug transport to the brain increases.2. In the early phase of ischemia and reperfusion function protein and morphosis of BBB change to the negative direction together, and the former changes faster. At this time the permeability of BBB begins to increase. In the metaphase both sides change to the opposite direction. Although tissue structure does not appear the obvious reparation response function protein has already unceasingly increase. But newly synthetical protein is hard to make up the damage of structure barrier. So the permeability of BBB further increases. In the advanced phase both sides change to the positive direction together and that of BBB decreases. It can be seen that the alteration of ultramicrostructure and expression of P-glycoprotein in the state of ischemia and reperfusion have a close relation with the time of reperfusion. Both of them determine the drug transport to the brain in common. So it is the basic way to increase the drug concentration in the brain by effectively opening the drug transporters of BBB.3. Active principles of Qingkailing have the compatible effects on penetrating to BBB in normal and ischemia and reperfusion rats, and each component lays particular emphasis on action target. Baicalin causes the enhancement of permeability of BBB, which is helpful for the permeation of jasminoidin. Jasminoidin may not only lessen the damage of CMEC but also protect the damaged neurons by penetrating BBB. But taurocholic acid can enhance the expression of P-glycoprotein to excrete noxious substance and make up the damage of structure barrier. Qingkailing can obviously relieve brain edema. So this effect reveals the action mechanism of relieving the toxin and dredging the collateral of Qingkailing. It can provide a more effective way for study the drugs curing encephalopathy.4. The complex prescription of Chinese medicine curing encephalopathy has the favourable effect connected with the influence of active principles of Qingkailing on the morphosis and function of BBB. Compatible application of Chinese medicine educes the protection of poly-links and poly-targets. It can not only improve and restructurate the damaged BBB but also specially open BBB by influencing drug transporters, and increase the concentration of drugs into the brain and improve the function of cerebral nerves. This mechanism of action may just elucidate the key element of Chinese medicine curing encephalopathy.