Combination of Ligusticum chuanxiong and Radix Paeoniae ameliorate focal cerebral ischemic in MCAO rats via endoplasmic reticulum stress-dependent apoptotic signaling pathway.
BACKGROUND
Combination of Ligusticum chuanxiong and Radix Paeoniae (XS) is highly effective in the treatment for focal cerebral ischemic, but the underlying mechanism is not clear. This study was conducted to evaluate the combinative effects of XS on MCAO rats and explore the underlying mechanisms.


METHODS
MCAO rats were used to evaluate the protective effect of Ligusticum chuanxiong (CX), Radix Paeoniae Rubra (CS) and their combination (XS) on ameliorating focal cerebral ischemic. Cerebral ischemia deficits and infarct size were performed by 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin (H-E) staining. Activities of SOD, CAT and GSH-Px, as well as levels of LPO and MDA were detected by commercial kits while ELISA kits for the content of plasminogen activator inhibitor-1 (PAI-1) and plasminogen activator (PA). Immunohistochemistry (IHC) and western blot analysis (WB) were carried out to examine the protein expressions including PKR-like endoplasmic reticulum kinase (PERK), cytoplasmic of glucose regulated protein 78 (GRP78), X box-binding protein-1 (XBP-1), activating transcription factor-6 (ATF-6), C/EBP-homologous protein (CHOP), metalloprotease-9 (MMP-9), tissue inhibitor of metalloproteinase-1 (TIMP-1), Bcl-2 associated X protein (Bax), and porcineB-cellleukemia/lymphoma-2 (Bcl-2) in brain tissues. Reverse transcription polymerase chain reaction (RT-PCR) and Quantitative PCR (Q-PCR) were applied to examine vascular endothelial growth factor (VEGF) and N-methyl-d-aspartate receptors (NMDAR1) mRNA levels.


RESULTS
CX, CS and their combination (XS) could reduce cerebral ischemia deficits and infarct size of MCAO rats. They increased SOD, CAT and GSH-Px activities, and reduced MDA and LPO levels in serum, markedly. A significant decrease of endoplasmic reticulum stress-related factors PERK, XBP-1, ATF-6 and CHOP protein expression levels while an increase of GRP78 and MVD expression by the treatment of CX, CS and XS. It could also be observed that their treatment could reduce apoptotic damage of brain tissues by up-regulating Bax level and down-regulating Bcl-2 level. Furthermore, the levels of MMP-9 and PAI-1 in serum and tissues of rats were down-regulated remarkably while TIMP-1 and PA levels were up-regulated. VEGF mRNA level was up-regulated dramatically whereas NMDAR1 was reduced. Importantly, the combination of CX and CS, namely XS, has a more meaningful improvement on focal cerebral ischemic than CX or CS alone.


CONCLUSIONS
All these revealed that the combined XS exerted more remarkable protective effects than alone. XS could inhibit neuronal apoptosis by attenuating ER-stress-dependent apoptotic signaling and protected the blood-brain barrier. These findings might supply beneficial hints for the synergy of CX and CS, and provide the basis for rationality of XS preparation and deserve further clinical investigations.
Jiangsu Collaborative Innovation Center of Chinese Medicinal Resources Industrialization, National and Local Collaborative Engineering Center of Chinese Medicinal Resources Industrialization and Formulae Innovative Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210023, PR China; Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, PR China; State Key Laboratory Breeding Base of Dao-di Herbs, China Academy of Chinese Medical Sciences, Beijing, 100700, PR China; Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu 210028, PR China.
Junfei
J
Gu
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, PR China; Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu 210028, PR China.
Juan
J
Chen
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, PR China; Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu 210028, PR China.
Nan
N
Yang
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, PR China; Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu 210028, PR China.
Xuefeng
X
Hou
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, PR China; Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu 210028, PR China.
Jing
J
Wang
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, PR China; Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu 210028, PR China.
Xiaobin
X
Tan
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, PR China; State Key Laboratory Breeding Base of Dao-di Herbs, China Academy of Chinese Medical Sciences, Beijing, 100700, PR China; Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu 210028, PR China. Electronic address: wenmoxiushi@163.com.
Liang
L
Feng
Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine, Nanjing, Jiangsu 210028, PR China; Key Laboratory of New Drug Delivery System of Chinese Materia Medica, Jiangsu Provincial Academy of Chinese Medicine, Nanjing, Jiangsu 210028, PR China. Electronic address: xiaobinjia_nj@126.com.
Xiaobin
X
Jia
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D001921:Brain / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002374:Catalase / Q000378:metabolism; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D005978:Glutathione / Q000378:metabolism; D005979:Glutathione Peroxidase / Q000378:metabolism; D020244:Infarction, Middle Cerebral Artery / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D030004:Ligusticum; D008297:Male; D008315:Malondialdehyde / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D018696:Neuroprotective Agents / Q000494:pharmacology / Q000627:therapeutic use*; D029596:Paeonia; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism; D010960:Plasminogen Activators / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D016194:Receptors, N-Methyl-D-Aspartate / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D013482:Superoxide Dismutase / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D002374:Catalase; D005978:Glutathione; D005979:Glutathione Peroxidase; D008315:Malondialdehyde; D020780:Matrix Metalloproteinase 9; D018696:Neuroprotective Agents; D017395:Plasminogen Activator Inhibitor 1; D010960:Plasminogen Activators; D019253:Proto-Oncogene Proteins c-bcl-2; D012333:RNA, Messenger; D016194:Receptors, N-Methyl-D-Aspartate; D013482:Superoxide Dismutase; D019715:Tissue Inhibitor of Metalloproteinase-1; D042461:Vascular Endothelial Growth Factor A; D051028:bcl-2-Associated X Protein; C494828:Bax protein, rat; C576839:TIMP1 protein, rat; C467498:vascular endothelial growth factor A, rat; C572337:Mmp9 protein, rat
Albiflorin (PubChem CID: 51346141); Benzoyl paeoniflorin (PubChem CID: 73157327); Blood-brain barrier; Caffeic acid (PubChem CID: 689043); Catechin (PubChem CID: 73160); Combination of Ligusticum Chuanxiong-Radix Paeoniae; ER-stress-dependent apoptotic signaling pathway; Ferulic acid (PubChem CID: 445858); Focal cerebral ischemic; Neuronal apoptosis; Oxypaeoniflorin (PubChem CID: 46882883); Paeoniflorin (PubChem CID: 75124163); Protocatechuic (PubChem CID: 72)