Effect Of Cleansing Qingkailing On OGD-Rep Injury Of SH-SY5Y Cells And Its Mechanism

Qingkailing Injection(QKL) was initally developed on the basis of Angong Niuhuang Bolus, which can clear heat and toxic materials to inducing resuscitation. QKL used mainly in the treatment of acute stroke, besides, it is also widely used in treating upper respiratory inffcection, pneumonia, hepatitis, toxicosis, tumour and infectious disease, etc., which involved in various clinical departments. With the increased use, more and more ADR of QKL are reported, which brings hidden danger for the health of majority of patients. To solve the problem, the relevant team of BUCM have developed new formulation of QKL, refined Qingkailing Injection(JZQKL), which chosed effective components from QKL so as to reduce side effects. Animal experiment showed that JZQKL possess remarkable anti-ischemia effect that equivalent to QKL. As the study progressed, new detection improved, perhaps we need to look again at the reasonability of the formula of JZQKL. In the project, we developed an ischemic model in SH-SH5Y cell line with treatment of oxyge glucose deprivation-repfusion(OGD-Rep), and investigated the protective effect of JZQKL on this OGD-Rep model.The first section of this article systematically review the clinical effects and safety of Qingkailing injection for the treatment of actue stroke. The second section is cells experiments, which is composed of the following three parts:1. To produce OGD-Rep model in SH-SY5Y cell line; 2. Pharmacodynamics study. To test and selcet protective drugs from JZQKL through CCK-8 assay, SRB staining and LDH assay, and try to set a new combination based on the results of the previous stage; 3. Mechanism research (oxidative stress, ER stress and autophagy)1 Objective1.1 To compare the drug action of QKL ans JZQKL on SH-SY5Y cell uder OGD-Rep model.1.2 To confirm the effects of BAL, Gen, HDCA, CA and TUDCA on the OGD-Rep model, as well as their dosage range.1.3 To adjust formulation of JZQKL, to find the best combination, and to validate its pharmacodynamics.1.4 To study that if the mechanisms of the new combination are concerned with oxidative stress, ER stress or autophagy.2 Method2.1 Model makingThe OGD-Rep Model was made by Anaeropouch combined with Glucose-free DMEM. OGD12h-Rep12h was selected as the main time, and cells damaged rate was about 50 percent.2.2 Pharmacology①Methods Cell growth rate was determined by CCK8, SRB assay, Damaged rate was detected by LDH assay kit.②The cytoprotection of the new combination(GT) was determined by Annexin V-FITC/PI assasy kit, which could show the necrosis and apoptosis rates of cells.2.3 Mechanism①The intracellular levels of reactive oxygen species (ROS) was detected by DCFH-DA (fluorescence probe).②Autophagosome or autolysosome were observed through AO (acridine orange)stain, MDC stain and Lyso-Tracker stain, to understand if the protective effect of GT is concerned with autophage.③The related proteins of ER stress and autophage, such as,Bip/GRP78, CHOP, LC3B, p62 were detected with western blot analysis to observe the GT combination whether produce protective effect by affecting autophage through regulating ER stress.3 Result3.1 Model makingOGD12h-Rep12h was selected as the main time, and cells damaged rate was about 50 percent.3.2 IC50 Detecting①The IC50 of QKL and JZQKL could not be obtained(maximum concentration 1000nL/mL). QKL showed significant damage at 5000 to 10000 nL/mL after 24h intervention. JZQKL significant inhibitory effect on the proliferation of the cells.②The IC50 of Gen could not be obtained(maximum concentration 2000μg/mL). After 24h intervent with Gen, the cell activity did not decrease, but showed a certain increase in the low dosages(CCK8 and SRB assay).③The IC50 values of BAL was 114.073μg/mL,95% CI (71.837,184.264) (CCK-8).The cell injury was positively correlated to the concerntration of BAL. Obvious cell loss was detected at 100μg/mL and above by SRB assay.④The IC50 values of HDCA was 502.989μg/mL,95%CI(315.465,1060.9)(CCK-8). The CCK-8 results showed significant inhibitory effect on the proliferation of the cells at 15.63μg/mL and above of HDCA. Obvious cell loss was detected at 125μg/mL and above by SRB assay.⑤The IC50 values of CA was 4.66μg/mL,95% CI (1.229,12.725) (CCK-8). The CCK-8 results showed significant inhibitory effect on the proliferation of the cells at 7.8μ g/mL and above of HDCA.⑥The IC50 values of TUDCA was 1955.693μg/mL,95%CI (1454.846,3147.317) (CCK-8). The CCK-8 results showed a decreased tendency of the proliferation of the cells at 500μg/mL, and showed significant inhibitory effect at 1000μg/mL.3.3 Protective effect①Under OGD12h-Rep2h model(moderate damage), QKL showed a slight decrease in cell vitality at 6.3～50nL/mL, while JZQKL showed definite protective effect at 12.5-100nL/mL.Under OGD18h-Rep2h model(severe damage), QKL and JZQKL showed damage effect starting at 25 nL/mL②Gen showed protective effect on OGD-Rep injured cells, and had no obvious toxic effects at 250-500μg/mL, whether on moderate or severe damaged model. There was no definite dose-dependent effect.③BAL showed obvious toxicity on injured cells, especially at 44μg/mL and above. The damage effect was dose dependent.④HDCA and CA showed certain cytotoxicity respectively at 20-60μg/mL and 300 u g/mL and above, while TUDCA was 500μg/mL3.4 Effect of the combination①Gen+BAL When Gen(62.5μg/mL) combined respectively with BAL 0.55,5.5,11 μg/mL, the cell viability decreased with the rise in BAL concertration(CCK-8), and the cell nubmer vice versa(SRB).②Gen+HDCA When Gen(62.5μg/mL) combined respectively with HDCA 5,10μ g/mL, the cell viability showed certain decrease(difference was not statistically significant).③Gen+CA When Gen(100μg/mL) combined respectively with CA5,10,25,50μ g/mL, the cell viability decreased with the rise in CA concertration(CCK-8).④Gen+TUDCA There no obvious damage effect when Gen combined with TUDCA. The cell viability had slightly increase at 0.5 and 5μg/mL of TUDCA.3.5 Efficacy confirmingThe results of CCK-8 and SRB assays showed that TUDCA(5μg/mL) could slightly promote the protective effect of Gen, but there was no statistical significance, and TUDCA(50-100μg/mL or above) could weaken the protective effect of Gen. Flow cytometry showed that TUDCA (5μg/mL) combinede with Gen(100μg/mL) had lower apoptosis rate than Gen(100μg/mL) alone.3.6 Mechanism study①Oxidative Stress The detection of active oxygen species:the level of ROS increased after OGD-Rep injury, and the fluorescence intensity of GT group was weaker than that of model group.②ER stress The influence of the regulators of ER stress Tm and SAL on drug efficacy of GT:All the three groups GT, Tm and Tm+GT had some effects on improving the cell vitality, and there were no statistically significant difference among the three groups. SAL had no obvious effect on the cell vitality, but decreased the efficacy of GT.③Autophagy Fluorescence staining of autophagosome(or autolysosome):A. AO stain, Red fluorescence of the model and drug groups were darker than the control group, and the model group was the darkest, next GT+Tm, and the differences were not statistically between GT and GT+SAL group. B. MDC stain, Green fluorescence of control group was in disperse state, while that of model accumulated perinuclear, and there were no statistically significant difference among the other groups. C. Lyso-Traker, The concentrations of red fluorescence of control group was more than the other groups, but there were no obviously difference among the rest groups.4 Conclusion4.1 Comparison of QKL and JZQKL①For normal cells, JZQKL had less toxicity than QKL.②For injured cells, JZQKL had less toxicity and more protective effect than QKL.4.2 IC50 Detecting①The IC50 of QKL and JZQKL could not be obtained(maximum concentration 1000nL/mL), but their showed significant damage at 5000 to 10000 nL/mL after 24h intervention.②The IC50 of Gen could not be obtained(maximum concentration 2000μg/mL).③The IC50 of BAL was 114.073μg/mL,95% CI (71.837,184.264) (CCK-8).④The IC50 of HDCA was 502.989μg/mL,95%CI (315.465,1060.9) (CCK-8).⑤The IC50 of CA was 4.66μg/mL,95%CI (1.229,12.725) (CCK-8).⑥The IC50 of TUDCA was1955.693μg/mL,95%CI(1454.846,3147.317)(CCK-8). 4.3 Protective effect①Under moderate damage model, JZQKL showed certain protective effect, and better than QKL. Under severe damage model, QKL and JZQKL, neither of them had protective effect.②Gen showed protective effect on OGD-Rep injured cells. There was no definite dose-dependent effect.③BAL showed obvious toxicity on injured cells, especially at 44μg/mL and above. The damage effect was dose dependent.④HDCA and CA showed certain cytotoxicity respectively at 20-60μg/mL and 300 u g/mL and above, while TUDCA was 500μg/mL.4.4 Effect of the combination①When Gen combined respectively with BAL, HDCA and CA, the cell viability decreased with the rise in their concertration.②TUDCA(50-100μg/mL or above) could weaken the protective effect of Gen. Flow Low concerntration of TUDCA (5μg/mL) could improve the efficacy of Gen in apoptosis rate.4.5 Mechanism study①OGD-Rep injure might induce oxidative stress in SH-SY5Y cells, and lead to oxidized trauma. GT might afford protective effect through inhibitiong oxidative stress.②OGD-Rep injure might induce ER stress in SH-SY5Y cells. GT might play a role in protecting injured cells through regulating ER stress.③OGD-Rep injure might induce autophagy in SH-SY5Y cells. GT might play a role in protecting injured cells through regulating the early formation of autophage organelles.