[1] 9w Shenfu injection attenuates neonatal hypoxic-ischemic brain damage in rat

[1] 202w Perinatal hypoxia-ischemia remains the most important cause of brain injury in the newborn. However, there is still no effective cure for neonatal hypoxic-ischemic brain damage (HIBD). In the present study, we aimed to examine the neuroprotective effects of Shenfu injection (SFI) on HIBD of neonatal rat. Sprague-Dawley rats were divided randomly into three groups (n = 8): S group: the rats were sham operated; C group: the rats were operated for HIBD modeling and received intraperitoneal injection of saline; SFI group: the rats were operated for HIBD modeling and received intraperitoneal injection of SFI (10 ml/kg days) for 7 days. Flow cytometry analysis showed that apoptosis rate of neuron in hippocampal CAI region in SFI group was significantly less than in NC group at 3 and 7 days after HI insult (P \ 0.05). Immunohistochemical staining demonstrated that Bcl-2 expression was markedly higher while Bax expression was significantly lower in SFI group than in the C group at 24, 72 h and 7 days after HI insult (P \ 0.05). Our findings suggest that SFI exhibits neuroprotective effects for neonatal hypoxicischemic brain injury by preventing neuron apoptosis and has potential to be used in the clinical for the treatment of perinatal hypoxia-ischemia.

[1] 54w Perinatal hypoxia-ischemia remains the most important cause of brain injury in the newborn, leading to death or lifelong sequelae. White matter injuries in newborn infants have long-term effects on physical, visual, motor, sensory, cognitive and social development in human infants [1]. However, there is still no effective cure for neonatal hypoxic-ischemic brain damage (HIBD).

[2] 91w Shenfu injection (SFI) is an extract of traditional Chinese herbs that mainly consists of ginsenoside ginsenoside Rb1 and aconitine alkaloids. SFI has been shown to exhibit protective effects against ischemia injuries on a variety of organs such as the brain, lung, kidney, intestines, liver and heart [2][3][4]. To date, most of the studies on the mode of action of SFI have focused on its protective effects on cardiacmyocytes. In the present study, we aimed to examine the neuroprotective effects of SFI on HIBD of neonatal rat and investigate the underlying mechanism.

[3] 93w It is known that hypoxic-ischemic brain injury is closely linked with the lipid peroxidation and the occurrence of apoptosis, and blocking neuronal apoptosis help reduce hypoxia-ischemic brain injury [5,6]. Several experiments showed that ginsenosides could inhibit ischemia-reperfusion injury-induced apoptosis of neurons [7][8][9]. Therefore, we speculate that SFI exhibits protective effects on HIBD in neonatal rats by inhibiting neuron apoptosis. In the present study, we found that SFI significantly inhibited apoptosis of hippocampus neurons during HIBD and this was associated with the upregulation of anti-apoptotic factor Bcl-2 and the downregulation of pro-apoptotic factor Bax.

[1] 96w To investigate the mechanism by which SFI reduces HIinduced neuron apoptosis, we examined the expression of apoptosis-related proteins in hippocampal CA1 region of rat. The expression of Bcl-2 in hippocampal CA1 region in SFI group and C group was increased at 2 h after HI insult, compared to S group, reached the peak at 24 h after HI insult, and gradually decreased at 3 days after HI. Notably, the expression of Bcl-2 in SFI group was markedly higher than in C group at 24, 72 h and 7 days after HI insult (Fig. 1; Table 2).

[2] 59w In contrast, the expression of Bax in hippocampal CA1 region in SFI group was significantly lower than in C group at 24, 72 h and 7 days after HI insult (Fig. 2; Table 3). These data suggest that SFI promotes the upregulation of Bcl-2 expression and the downregulation of Bax expression in hippocampal CA1 region of mice after HI.

[1] 270w The present data indicate that SFI is an effective neuroprotective agent for neonatal rats subjected to hypoxicischemic insult. As a model of human brain development, the post-natal day 7 (P7) mouse or rat approximates a 34-36 week premature human [11]. This developmental age was used because, for either rats or mice, this age falls in the middle of the brain growth spurt period. HI injury in the P7 rat and P7 mouse produces deficits, defined as performance deficits on spatial memory tasks, and impaired motor performance, defined as decreased ability to run on a rotating rod and circling following apomorphine, but not abnormal locomotor activity in adult animals [12,13]. Although functional parameters were not measured in this study, our data provide evidence that SFI has a strong neuroprotective effect, and therefore, may improve functional outcomes. We found that SFI decreases neuronal damage and that the protective effects of SFI are associated with its ability to reduce apoptotic cell death in the neonatal rat brain. These results are consistent with a influx to the mitochondrial membrane depolarization and improve the energy metabolism of nerve cells [17]. In this study we demonstrated SFI plays a role in the promotion of neuron survival and prevention of neuron apoptosis via the upregulation of anti-apoptotic gene Bcl-2 and the downregulation of pro-apoptotic gene Bax. Although further studies are necessary to understand how SFI modulates the expression of apoptosis-related proteins in neurons subjected to HI, our findings suggest that SFI exhibits beneficial effects for neonatal hypoxic-ischemic brain injury and has the potential to be used in the clinical for the prevention and treatment of perinatal hypoxia-ischemia.

[1] 137w Shenfu was produced by Ya'an 39 Pharmaceutical Co., Ltd (Batch No. 991210). The main components of Shenfu injection included ginsenoside ([0.8 mg/m1) and aconitine (\0.1 mg/m1). The postnatal 7 day Sprague-Dawley (SD) rats (both male and female, 12-16 g) were provided by Experimental Animal Center of Xuzhou Medical College. The rats were randomly divided into three groups (n = 8): S group: the rats were sham operated; C group: the rats were operated for HIBD modeling and received intraperitoneal injection of saline; SFI group: the rats were operated for HIBD modeling and received intraperitoneal injection of SFI (10 ml/kg days) for 7 days. HIBD modeling was performed as described previously with the pups undergoing the ligation of the right common carotid artery [10]. In sham-operated pups, the left common carotid artery was exposed, but was not ligated.

[2] 164w The brain specimens were immediately fixed with 10 % formaldehyde, and cut as 4 lm sections. The sections were washed carefully with 0.01 M phosphate buffered saline (PBS) three times. To block endogenous peroxidases, the sections were treated with 3 % hydrogen peroxide for 20 min. The sections were blocked with 2 % goat serum in 0.01 M PBS containing 0.3 % Triton X-100 for 1 h at room temperature, then incubated at 4 °C overnight with antibody against Bcl-2 (1:250 dilution, Zhongshan, Beijing, China) or Bax (1:200 dilution, Zhongshan, Beijing, China). Afterwards, the sections were subjected to immunohistochemical staining using PV6000 kit (Zhongshan, Beijing, China). Finally the sections were developed with DAB and assessed under light microscopy. For negative controls, the primary antibodies were replaced with PBS. Four fields from each section were randomly selected, and the immunohistochemical staining score was calculated based on the average density of the positively stained areas using BioQuant NOVA Prime imaging analysis software (BioQuant, Nashville, TN, USA).

[3] 36w The data were expressed as the mean ± standard deviation (SD). Statistical comparison between the groups was performed by t test using SPSS 11.0 software and a P value less than 0.05 was considered statistical significance.

[1] 77w At different time points after HIBD, the rats were decapitated. The hippocampus was taken and resuspended in PBS, shredded and filtered through 200 mesh nylon net two times to make single cell suspension. The cell suspension was fixed in 70 % ethanol at 4 °C for 12 h, then stained by propidium iodide at 4 °C for 30 min in the dark. The samples were immediately analyzed by FACS and the data were analyzed by CellQuest software.

[2] 84w Compared to group S, neuron apoptosis rate was increased in hippocampal CA1 region in SFI group and C group 2 h after HI insult, reached the peak at 3 days, then gradually decreased and recovered to normal at 14 days. However, the increase of neuron apoptosis at 3 and 7 days after HI insult was significantly less in SFI group than in C group (P \ 0.05) (Table 1). These data indicate that SFI reduces HI-induced neuron apoptosis in hippocampal CA1 region of rat.