Mechanism Of High-dose Chlorogenic Acid And Its Chinese Herbal Injection-induced Microcirculation Disturbance

Objectives:Chlorogenic acid (CA) exits widely in those Chinese herbal injections that have antibacterial and antiphlogistic effects and belongs to the ethnopharmacological family of medicines. Chinese herbal injections containing high levels of CA have been reported to increase the adverse drug reactions, but the mechanism for which is still unclear. In this study, we investigated the potential role of CA, Shuang Huang Lian injection (SHL) and other phenolic acids in initiating microcirculation disturbance and the underlying mechanisms.Methods:Male Wistar rats were treated with different dosages of CA, SHL or other phenolic acids (caffeic acid, salvianolic acid B, and ferulic acid) for different time period. Examined were microcirculation by intravital microscopy, histology of ileum tissue, expression of adhesion molecules CDllb and CD18on leukocytes by flow cytometry, myeloperoxidase activity and maleic dialdehyde content in ileum tissue by spectrophotometry, activity of superoxide dismutase, catalase, TNF-α, IL-6, IL-10, ET-1and iNOS in serum by ELISA, and expression of NADPH oxidase subunits N0X4, p22phox,p47phox and nuclear factor kappa B by Real-time PCR and/or Western blot.Results:The present study shown that intravenous injection with CA may cause a range of inflammatory reactions, manifested as increased expression of adhesion molecules CDllb and CD18on leukocytes, leukocyte recruitment in microvessels and inflammatory cell infiltration, microvessel hyperpermeability, increased level of IL-6and TNF-α in serum. Moreover, exposure to CA brought about an imbalance between oxidant and antioxidant mechanisms, leading to an oxidant stress, including increased production of ROS in venular wall, enhanced activities of MDA and MPO, and decreased activities of SOD and CAT. The expression of N0X4, p22phox and p47phox was observed being increase in response to CA challenge, indicating the involvement of NADPH oxidase in the CA-induced oxidant stress. On the other hand, CA had no effect on mast cell degranulation suggesting that the mast cell has not been triggered. Noticeably, all the observed detrimental effects occurred only after addition of high dose, but not low-dose or middle-dose of CA.Injection with high-dose SHL may also cause a range of imbalance between oxidant and antioxidant mechanism, leading to an oxidant stress, including increased production of ROS in venular wall, and decreased activity of CAT. The expression of N0X4and p47phox was observed being increase in response to high-dose SHL injection challenge, indicating the involvement of NADPH oxidase in the SHL-induced oxidant stress. Moreover, exposure to high-dose CA or SHL undermined the balance of basal levels of pro-(TNF-a) and anti-(IL-10) inflammatory cytokines, manifested as decreased IL-10to TNF-a ratio. Meanwhile, activation of the critical transcriptional factor NF-κB, which involved in proinf lammatory mediator production, was increased in certain extent within different tissues. SHL also had no effect on mast cell degranulation, which suggested that the mast cell has not been triggered and the allergenicity played no part in our observations. Noticeably, all the observed detrimental effects occurred only after addition of overdose SHL, but not routine dose.The study also found that high-dose FA brought about an excessive production of ROS in venular walls, and made the increasing expressions of N0X4. These effects were similar to the potential role of high-dose CA.Conclusions:The study documented that:1) High-dose CA generated peroxide in vascular endothelial cells through NADPH oxidase, which causes enhanced expression of adhesion molecules, including CDllb/CD18on leukocytes, leading to leukocyte rolling and adherence to postcapillary venules. In addition, leukocytes adherent to postcapillary venules provoke the release of oxygen free radicals, inflammatory factors and albumin leakage. The ROS induce lipid peroxidation and accompanied increased permeability of venules which exacerbates subsequent injuries to endothelial cells and basement membrane. It is different from typical allergic reactions since mast cell does not take part in this process.2) Overdose SHL injection was similarly induced excessive ROS in vascular endothelial cells through NADPH oxidase and imbalance of basal levels of pro-(TNF-α) and anti-(IL-10) inflammatory cytokines.3) High-dose FA provoked an excessive ROS production from venules, which was also similar to high-dose CA. In summary, the present study showed that intraveinal infusion of CA provoked oxidative stress and inflammatory reaction if administrated at a high-dose, suggesting the double-edged sword property of CA as a drug. These results provide new insight for better understanding the mechanism underlying the adverse effect of CA, and alarm the clinician to be cautious especially when a Chinese herbal injection containing high level of CA is to be applied.