| Objective:1. To explore the neuroprotection of xuesaitong injection and NEPl-40 on the expression of NgRl, RhoA and ROCK Ⅱ in Sprague-Dawley (SD) rats with cerebral infarction at different times and after SH-SY5Y cells suffering the injury with oxygen-glucose deprivation. 2. To explore the influence of xuesaitong injection and NEPl-40 on the expression of pro-inflammatory cytokines IL-1β, TNF-α and anti-inflammatory cytokines IL-10, TGF-β1 in serum and infarcted cortex 7 days after cerebral infarction in SD rats.Methods:I. Firstly, the improved surgery of middle cerebral artery occlusion(MCAO) were applied to mimic the model of cerebral infarction, using the male SD rats with SPF level. The effect of MCAO surgery were evaluated by TTC staining, after improving the achievement ratio of operation by means of experimental practice.2. The SD rats with SPF level were randomly divided into the sham-operation, MCAO model, MCAO with xuesaitong treatment, MCAO with NEP1-40 treatment and MCAO with xuesaitong plus NEPl-40 treatment groups. All of the rats were performed the MCAO surgery except for the sham group, and each groups had 6 rats. After surgery, the different groups were administrated different drugs with recording the body mass and neural deficit score(NDS) everyday. The samples were collected at 7 days,14 days and 28 days after surgery to detect the protein expression of NgRl, RhoA and ROCK Ⅱ in the infarcted cortex using the methods of immunohistochemistry (IHC) and western blot. On the other hand, the cells model were also established by using SH-SY5Y suffered the injury with oxygen-glucose deprivation/reoxygenation (OGD/R), and the best drug concentration of xuesaitong and NEPl-40 were explored by CCK8. Afterwards, the SH-SY5Y cells were divided into the normal, model, xuesaitong, NEP1-40 and xuesaitong plus NEP1-40 groups, with the normal group not performing the OGR/R injury. The cell viability were detected by using the method of CCK8 after the OGD/R injury, the protein and mRNA expression of NgRl,RhoA and ROCK Ⅱ were performed by western blot and quantitative real-time PCR(qRT-PCR).3. The SD rats with SPF level were randomly divided into the sham-operation, MCAO model, MCAO with xuesaitong treatment, MCAO with nimodipine treatment, MCAO with NEP1-40 treatment and MCAO with xuesaitong plus NEP1-40 treatment groups. All of the rats were performed the MCAO surgery except for the sham group, and each groups had 6 rats. The six groups were treated with different drugs after surgery, and the samples were collected at day 8 through drawing blood from abdominal aorta and brain tissue after decollation. The expression of IL-1β TNF-α and IL-10, TGF-β1 were detected by ELISA in serum and by IHC, western blot and qRT-PCR in the infarcted cortex.Results:1. TTC staining showed that sections from the MCAO rat had extensive pale areas whereas the sham-operated rat stained red, indicating the cerebral infarction model in rat were successful through the improved MCAO surgery. Meanwhile, our research group mastered the MCAO surgery and enhanced the success rate of surgery.2. The body mass in the sham group were not obvious change after surgery, and growing as the times went on. The body mass in the model group 7,14 and 28 days after surgery were significantly reduced when compared with the sham group (P<0.01). The body mass in the treated group including xuesaitong, NEPl-40 and combined drugs groups were slow increased 7 and 14 days after surgery, but elevated significantly as compared to the model group (P<0.01 or P<0.05). The body mass in the surgery group 28 days after surgery were increased markedly, and the treated group’s body mass were elevated significantly when compared with the model group (P<0.01). On the other hand, there were no neurologic impairment in the sham group. The neural deficit scores (NDS) 7 days after surgery in the model group were increased markedly when compared with the sham group (P<0.01), and the NDS in the treated group were reduced significantly when compared with the model group (P<0.01). The NDS in the model group on day 14 and 28 after surgery were increased markedly when compared to the sham group (P<0.01), the NDS in the treated group were reduced but with no statistical differences (P>0.05).3. The protein expression of NgRl, RhoA and ROCK Ⅱ7 days after surgery in the model group were elevated markedly when compared with the sham group (P<0.01). The expression of NgRland RhoA in the NEP1-40 group were reduced significantly when compared with the model group, the expression of ROCK Ⅱ was also reduced with no statistical difference (P>0.05). The expression of NgRl, RhoA and ROCK Ⅱ in the NEP1-40 and xuesaitong plus NEP1-40 groups were reduced significantly when compared with the model group in the infarcted cortex (P<0.01 or P<0.05).4. The protein expression of NgRl, RhoA and ROCK Ⅱ14 days after surgery in the model group were elevated markedly when compared with the sham group (P<0.01). The expression of NgRl, RhoA and ROCK Ⅱ in the xuesaitong and NEP1-40 groups were reduced significantly when compared with the model group in the infarcted cortex (P<0.01 or P<0.05). The expression of NgR1 and ROCK Ⅱin the xuesaitong plus NEPl-40 group were reduced markedly (P<0.01 or P<0.05), and the expression of RhoA was reduced significantly in western blot (P<0.01), but with no statistical difference in IHC.5. The protein expression of NgRl, RhoA and ROCK Ⅱ 28 days after surgery in the model group were elevated markedly when compared with the sham group in the infarcted cortex (P<0.01 or P<0.05). The expression of NgRl, RhoA and ROCK Ⅱ in the xuesaitong, NEP1-40 and xuesaitong plus NEP1-40 groups were reduced markedly when compared with the model group (P<0.01 or P<0.05).6. In the cell experiment, the best drug concentration of NEP1-40 was 10 ng/mL in SH-SY5Y cells suffered the injury with oxygen-glucose deprivation/reoxygenation (OGD/R), and the best drug concentration of xuesaitong determined by CCK8 was 320 mg/mL.7. The cell viability in the model group was rduced markedly when compared with the normal group after OGD/R injury in SH-SY5Y (P<0.01), and the cell viability in the treated groups were elevated significantly when compared with the model group (P<0.01). Meanwhile, the protein and mRNA expression of NgRl, RhoA and ROCK Ⅱ in the model group were elevated obviously when compared with the normal group (P<0.01 or P<0.05). The protein and mRNA expression of NgR1, RhoA and ROCK Ⅱ in the NEP1-40, xuesaitong and xuesaitong plus NEP1-40 groups were reduced significantly when compared with the model group (P<0.01 or P<0.05).8. In the model group, the expression of IL-1β in serum was increased slightly with no statistical significance (P>0.05), and the expression of TNF-α in serum was elevated (P<0.01), the IL-10 was reduced (P<0.05), but the expression of TGF-β1 was increased (P<0.05) 7 days after cerebral infarction in SD rats. The xuesaitong could reduce the serum expression of IL-1β and TNF-α with no statistical significance (P>0.05), and elevate the serum expression of IL-10 (P<0.05) and TGF-β (P>0.05) when compared with the model group. The nimodipine could reduce the serum expression of TNF-α (P<0.05) and IL-1β (P<0.01) and elevate the serum expression of IL-10 and TGF-β1 with no statistical significance (P>0.05). The NEP1-40 could reduce the serum expression of IL-1β and TNF-α, and promote the expression of IL-10 and TGF-β1 with no statistical significance (P>0.05). The xuesaitong plus NEP1-40 could reduce the serum expression of IL-1β and TNF-α (P<0.05), promote the expression of IL-10 (P<0.05) and TGF-β1 (P>0.05) when compared to the model group.9. The protein expression of pro-inflammatory cytokines IL-1β, TNF-α in the model group were elevated markedly (P<0.01 or P<0.05), the anti-inflammatory cytokine IL-10 was reduced significantly (P<0.01 or P<0.05), whereas another anti-inflammatory cytokine TGF-β1 was elevated significantly (P<0.01 or P<0.05) 7 days after cerebral infarction in SD rats, when compared with the sham group. The xuesaitong could reduce the protein expression of IL-1β and TNF-a (P<0.01), and promote the expression of IL-10 (P<0.05), but have no obvious effect in the protein expression of TGF-β1, when compared with the model group. The nimodipine could reduce the protein expression of IL-1β and TNF-a (P<0.01), but have not statistical significance in the protein expression of IL-10 and TGF-β1 (P>0.05), when compared to the model group. The NEP1-40 could reduce the protein expression of EL-1β and TNF-a (P<0.01 or P<0.05), and promote the expression of IL-10 (P<0.05) and TGF-01 (P>0.05), when compared with the model group. The xuesaitong plus NEP1-40 could reduce the protein expression of IL-1β and TNF-a (P<0.01 or P<0.05), and promote the expression of IL-10 (P<0.05) and TGF-β1 (P>0.05), when compared with the model group.10. The mRNA expression of pro-inflammatory cytokines IL-1β, TNF-a in the model group were elevated markedly (P<0.01), the anti-inflammatory cytokine IL-10 was reduced significantly (P<0.05), whereas another anti-inflammatory cytokine TGF-β1 was increased exponentially (P<0.05) 7 days after cerebral infarction in SD rats, when compared with the sham group. The xuesaitong could reduce the mRNA expression of IL-1β and TNF-a (P<0.01), and promote the mRNA expression of IL-10 (P<0.01) and TGF-pl (P>0.05), when compared with the model group. The nimodipine could reduce the mRNA expression of IL-1(3 and TNF-a (P<0.01), suppress the mRNA expression of IL-10 (P>0.05) and facilitate the mRNA expression of TGF-β1 (P>0.05), when compared to the model group. The NEP1-40 could markedly reduce the mRNA expression of EL-1β and TNF-α (P<0.01), promote the mRNA expression of IL-10 (P<0.05), but have no obvious effect in the mRNA expression of TGF-β1 (P>0.05), when compared with the model group. The xuesaitong plus NEP1-40 could markedly reduce the mRNA expression of IL-1β and TNF-a (P<0.01), and promote the mRNA expression of IL-10 and TGF-β1 but with no statistical difference (P>0.05), when compared with the model group.Conclusions:1. The improved MCAO surgery using suture method could successfully mimic the human disease of focal cerebral infarction, and has a high success rate after practice.2. NEP1-40 could directly suppress the expression of NgRl, RhoA and ROCK Ⅱ, meanwhile regulate the expression of pro-inflammatory cytokines IL-1β, TNF-α and anti-inflammatory cytokines IL-10, TGF-β1, thus to accelerate the neural regeneration and functional recovery after cerebral infarction in SD rats.3. The xuesaitong injection plays a similar role as NEP1-40 via suppressing the expression of NgR1/RhoA/ROCK Ⅱ signaling pathway, meanwhile exerts the neuroimmunomodulatory role through inhibiting the pro-inflammatory cytokines IL-1β, TNF-α and facilitating the anti-inflammatory cytokine IL-10. The xuesaitong injection functions a comprehensive neuroprotection to accelerate the neural regeneration and functional recovery after cerebral infarction in SD rats.
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