| Evidence Sentence: |
Scalp Acupuncture and Treadmill Training Inhibits Neuronal Apoptosis through Activating cIAP1 in Cerebral Ischemia Rats |
| Evidence Sentence: |
Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and neuronal nuclear protein (NeuN) double staining and cellular inhibitor of apoptosis protein-1 (cIAP1) and NeuN immunofluorescence double staining were used to detect the short-term and long-term neuroprotective effects of scalp acupuncture combined with treadmill training on pMCAO rats. |
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In addition, the antiapoptotic effect of the combined treatment was evaluated in pMCAO rats transfected with cIAP1 shRNA. |
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Western blotting was used to detect the relative protein expression in the caspase-8/-9/-3 activation pathway downstream of cIAP1 to further clarify its regulatory mechanism. |
| Evidence Sentence: |
Our results showed that scalp acupuncture combined with treadmill training successfully achieved short-term and long-term functional improvement within 14 days after stroke, significantly inhibited neuronal apoptosis, and upregulated the expression of cIAP1 protein in the ischemic penumbra area of the ischemic brain. |
| Evidence Sentence: |
However, no significant functional improvement and antiapoptotic effect were found in pMCAO rats transfected with cIAP1 shRNA. |
| Evidence Sentence: |
These findings indicate that scalp acupuncture combined with treadmill training therapy may serve as a more effective alternative modality in the treatment of ischemic stroke, playing an antiapoptotic role by upregulating the expression of cIAP1 and inhibiting the activation of the caspase-8/-9/-3 pathway. |
| Evidence Sentence: |
Effect of SA-T on cIAP1 Expression in Neurons of the pMCAO Model |
| Evidence Sentence: |
Apoptosis is a critical factor for the loss of hypoxic-ischemic neurons in the nervous system, and cIAP1 is an important regulator of apoptosis. |
| Evidence Sentence: |
The double immunofluorescence staining results showed that multitudinous cIAP1 and NeuN positive neurons existed in the cortex penumbra of the ischemic brain in the sham-operated group (Figure 4). |
| Evidence Sentence: |
Compared with the sham-operated group, the fluorescence intensity of cIAP1 and NeuN proteins and the number of cIAP1 and NeuN positive neurons were markedly reduced on the 3rd, 7th, and 14th days in the model group (P < 0.05). |
| Evidence Sentence: |
SA, TT, and SA-T significantly increased the fluorescence intensity of cIAP1, NeuN, and the number of cIAP1 and NeuN positive neurons in pMCAO rats (P < 0.05), with SA-T showing the highest effect. |
| Evidence Sentence: |
Effects of Three Pairs of shRNA Expression Vectors on cIAP1 Expression |
| Evidence Sentence: |
The results of Western blotting and RT-PCR showed that there was no decline in cIAP1 protein or mRNA expression in the negative group (NC) as compared with the control group. |
| Evidence Sentence: |
However, the expression of cIAP1 protein was decreased by 39%, 75%, and 25% in the shRNA-1, shRNA-2, and shRNA-3 groups, respectively (Figure 5(a)). |
| Evidence Sentence: |
Moreover, cIAP1 mRNA expression was decreased in the shRNA-1, shRNA-2, and shRNA-3 groups by ~30%, 75%, and 18%, respectively (Figure 5(b)). |
| Evidence Sentence: |
Effect of SA-T on Behavioral Outcomes in the cIAP1 shRNA Interference pMCAO Model |
| Evidence Sentence: |
As shown in Figures 6(a)-6(c), the SA-T + Lv cIAP1 NC group showed the same trend as SA-T in behavioral outcomes, and there was no difference between the two groups (P > 0.05). |
| Evidence Sentence: |
However, the therapeutic effect of SA-T on cerebral ischemia was completely reversed by Lv cIAP1. |
| Evidence Sentence: |
There was no significant difference between SA-T + Lv cIAP1 and model groups (P > 0.05), and behavioral outcomes in the SA-T + Lv cIAP1 group were significantly worse than in the SA-T group (P < 0.05). |
| Evidence Sentence: |
Effect of SA-T on Neuronal Damage in the cIAP1 shRNA Interference pMCAO Model |
| Evidence Sentence: |
SA-T and SA-T + Lv cIAP1 NC treatment markedly ameliorated these pathological abnormalities, but no significant difference was observed between the two groups (P > 0.05). |
| Evidence Sentence: |
Compared with the SA-T group, more neuronal necrosis existed in the SA-T + Lv cIAP1 group, and the difference was statistically significant (P < 0.05) (Figure 6(d)). |
| Evidence Sentence: |
Nissl staining showed a similar trend with HE staining; fewer Nissl bodies were observed in the SA-T + Lv cIAP1 group when compared with the SA-T group (P < 0.05) (Figure 6(e)). |
| Evidence Sentence: |
Effect of SA-T on Neuronal Apoptosis and NeuN Expression in the cIAP1 shRNA Interference pMCAO Model |
| Evidence Sentence: |
Neuronal apoptosis in the cIAP1 shRNA interference pMCAO model was further determined by a TUNEL assay. |
| Evidence Sentence: |
Treatment with SA-T alone or combined therapy with Lv cIAP1 NC partly diminished neuronal apoptosis induced by cerebral ischemia (Figure 7(a)). |
| Evidence Sentence: |
The same results were also confirmed by Western blotting, which showed that the expression level of NeuN protein in the SA-T and SA-T + Lv cIAP1 NC group significantly increased when compared with the model group (P < 0.05) (Figure 7(b)). |
| Evidence Sentence: |
However, in the SA-T + Lv cIAP1 group, SA-T showed no significant antiapoptotic effect, and the number of TUNEL positive cells was markedly higher than that in the SA-T group (P < 0.05). |
| Evidence Sentence: |
In addition, Western blotting results showed that the NeuN protein expression level in the SA-T + Lv cIAP1 group was significantly lower than that in the SA-T group (P < 0.05). |
| Evidence Sentence: |
Even on the 3rd and 7th days, the expression of NeuN protein in the SA-T + Lv cIAP1 group was significantly lower than that in the model group (P < 0.05). |
| Evidence Sentence: |
Effect of SA-T on cIAP1 Expression in the cIAP1 shRNA Interference pMCAO Model |
| Evidence Sentence: |
To further investigate the influence of SA-T on the expression patterns of cIAP1 in the cIAP1 shRNA interference pMCAO model, immunohistochemistry and Western blotting were used to compare cIAP1 protein levels in the five groups on the 3rd, 7th, and 14th days (Figures 7(c) and 7(d)). |
| Evidence Sentence: |
The results showed that the expression level of cIAP1 in the SA-T and SA-T + Lv cIAP1 NC groups was upregulated when compared with the model group (P < 0.05), while no difference was found between the two groups (P > 0.05). |
| Evidence Sentence: |
The expression level of cIAP1 protein in the SA-T + Lv cIAP1 group was significantly lower than that in the SA-T group (P < 0.05), but there was no difference between the SA-T + Lv cIAP1 and model groups (P > 0.05). |
| Evidence Sentence: |
This further indicates that SA-T mainly plays a neuroprotective role by targeting cIAP1. |
| Evidence Sentence: |
Effect of SA-T on the Caspase-8/-9/-3 Activation Pathway in the cIAP1 shRNA Interference pMCAO Model |
| Evidence Sentence: |
As shown in Figure 8, the Western blotting results showed that SA-T significantly attenuated pMCAO-induced caspase-8, caspase-9, and caspase-3 activation (P < 0.05), but no additional effect was observed in the combined therapy with Lv cIAP1 NC. |
| Evidence Sentence: |
Moreover, we found that SA-T had no significant reversal effect on the activation of caspase-8, caspase-9, and caspase-3 in the Lv cIAP1-transfected pMCAO model when compared with the model group (P > 0.05), while there were statistically significant increases as compared with the SA-T group (P < 0.05). |
