Panax notoginseng saponins provide neuroprotection by regulating NgR1/RhoA/ROCK2 pathway expression, in vitro and in vivo.
BACKGROUND
Panax notoginseng saponins (PNS) extracted from a traditional Chinese herbal medicine, Panax notoginseng (Burkill) F.H. Chen (Araliaceae), which has been extensively used in treating coronary heart disease, ischemic cerebrovascular disease and hemorrhagic disorders in China over hundreds of years.


OBJECTIVE
This study explored whether panax notoginseng saponins (PNS) provided neuroprotective effects by inhibiting the expressions of NgR1, RhoA, and ROCK2 following middle cerebral artery occlusion in rats and oxygen-glucose deprivation/reoxygenation (OGD/R) injury in SH-SY5Y cells.


METHODS
2,3,5-Triphenyltetrazolium chloride staining was used to determine successful middle cerebral artery occlusion establishment in sham-operated and operated Sprague-Dawley rats 1 day after injury. The rats were randomly separated into sham, model, NEP1-40, PNS, and NEP1-40 plus PNS (N+P) groups. After 7 days of treatment, body mass and neurological deficit scores were analyzed. Tissues were harvested and analyzed by hematoxylin-eosin staining and immunohistochemical analysis, western blotting, and quantitative real-time PCR (qRT-PCR). The optimal drug concentration of NEP1-40 and PNS on SH-SY5Y cells exposed to OGD/R injury was determined by CCK8 analysis. qRT-PCR was used to measure mRNA expression profiles of NgR1, RhoA, and ROCK2 in SH-SY5Y cells subjected to OGD/R.


RESULTS
The results showed that MCAO surgery successfully produced an infarct, and the PNS, NEP1-40, and N+P groups exhibited increased body mass and ameliorated neurological deficits compared with the model group. NEP1-40 treatment markedly reduced NgR1 and RhoA overexpression when compared to the model group, although there was no significant difference in ROCK2 expression. PNS and N+P treatment significantly decreased NgR1, RhoA, and ROCK2 overexpression compared with the model group. However, N+P treatment did not result in a synergistic effect, as assessed by immunohistochemistry, western blotting, and qRT-PCR. Following optimal administration of PNS (160μg/ml) and NEP1-40 (10ng/ml) on SH-SY5Y cells exposed to OGD/R injury, cell viability in the NEP1-40, PNS, and N+P groups significantly increased compared with the model group, as assessed by CCK8 analysis. Additionally, NgR1, RhoA, and ROCK2 mRNA expression profiles were significantly less in the NEP1-40, PNS, and N+P groups compared with the model group.


CONCLUSIONS
PNS provided neuroprotective effects in a rat model of cerebral ischemia and SH-SY5Y cells exposed to oxygen/glucose deprivation injury by inhibiting the overexpression of NgR1, RhoA, and ROCK2.
Key Laboratory of Chinese Internal Medicine of Educational Ministry and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China.
Xiaowei
X
Shi
Department of pediatrics, Beijing University of Chinese Medicine Third Affiliated Hospital, Beijing University of Chinese Medicine, Beijing, China.
Wenjing
W
Yu
Key Laboratory of Chinese Internal Medicine of Educational Ministry and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China.
Tiantian
T
Yang
Department of Rehabilitation, Beijing Children's Hospital, Capital Medical University, Beijing, China.
Wei
W
Liu
Key Laboratory of Chinese Internal Medicine of Educational Ministry and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China.
Yizhou
Y
Zhao
Key Laboratory of Chinese Internal Medicine of Educational Ministry and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China.
Yikun
Y
Sun
Key Laboratory of Chinese Internal Medicine of Educational Ministry and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China.
Limin
L
Chai
Key Laboratory of Chinese Internal Medicine of Educational Ministry and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China.
Yonghong
Y
Gao
Key Laboratory of Chinese Internal Medicine of Educational Ministry and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China.
Bin
B
Dong
Key Laboratory of Chinese Internal Medicine of Educational Ministry and Beijing, Dongzhimen Hospital, Beijing University of Chinese Medicine, Beijing, China. Electronic address: lingqunzh@vip.sina.com.
Lingqun
L
Zhu
D000818:Animals; D001921:Brain / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D004195:Disease Models, Animal; D015971:Gene Expression Regulation, Enzymologic; D005947:Glucose / Q000172:deficiency; D006801:Humans; D020244:Infarction, Middle Cerebral Artery / Q000201:enzymology / Q000235:genetics / Q000473:pathology / Q000517:prevention & control*; D008297:Male; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D018696:Neuroprotective Agents / Q000302:isolation & purification / Q000494:pharmacology*; D000070816:Nogo Receptor 1 / Q000235:genetics / Q000378:metabolism*; D053723:Panax notoginseng / Q000737:chemistry*; D008517:Phytotherapy; D010936:Plant Extracts / Q000302:isolation & purification / Q000494:pharmacology*; D010946:Plants, Medicinal; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D017207:Rats, Sprague-Dawley; D012503:Saponins / Q000302:isolation & purification / Q000494:pharmacology*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D054460:rho-Associated Kinases / Q000235:genetics / Q000378:metabolism*; D020742:rhoA GTP-Binding Protein / Q000235:genetics / Q000378:metabolism*
D018696:Neuroprotective Agents; D000070816:Nogo Receptor 1; D010936:Plant Extracts; D012333:RNA, Messenger; C486580:RTN4R protein, human; C486581:Rtn4r protein, rat; D012503:Saponins; C066737:RHOA protein, human; C515852:ROCK2 protein, human; C580818:ROCK2 protein, rat; D054460:rho-Associated Kinases; D020742:rhoA GTP-Binding Protein; D005947:Glucose
Cerebral infarction; Ginsengnoside Rb1 (PubChem CID: 9898279); Ginsengnoside Rg1 (PubChem CID: 441923); Ginsenoside Rd (PubChem CID: 24721561); Ginsenoside Re (PubChem CID: 73149); NEP1-40; NgR1; Notoginsengnoside R1 (PubChem CID: 441934); Panax notoginseng saponins; ROCK2; RhoA