Objective Ginsenoside Rg1 could increase the tolerance of neural hypoxia and ischemia under stress, and play an anti-apoptotic effect in hypoxia ischemia brain damage (HIBD). To investigate the effects of ginsenoside Rg1 on neural apoptosis and recovery of neurological function in neonatal rats with HIBD, and to explore the possible mechanism. Methods Fifty-four 10-day-old SD rats (weighing 16-22 g) were randomly allocated into sham-operation group (Sham group, n=6), HIBD model group (HIBD group, n=24), and ginsenoside Rg1 treatment group (Rg1 group, n=24). SDrats in HIBD group and Rg1 group were made the models of HIBD by l igation of the right common carotid artery (CCA) and subsequently hypoxic ventilation (8%O2 plus 92%N2) for 2.5 hours; and in Sham group, the right CCA was only exposed without l igation of CCA and hypoxic ventilation. Intraperitoneal injection of 0.1 mL normal sal ine (NS) containing 40 mg/kg Rg1 was given immediately after operation in Rg1 group, intraperitoneal injection of 0.1 mL pure NS was given in both HIBD group and Sham group and was repeated every 24 hours. The general state of SD rats was monitored after operation, and Longa scores were recorded to evaluate the neurological function at 4, 8, 24, and 72 hours after HIBD. Western blot and immunohistochemistry staining were used to detect protein expressions of both hypoxia inducible factor 1α (HIF-1α) and cleaved caspase 3 (CC3). TUNEL staining was used to evaluate neural apoptosis in situ. Results All rats survived to the end of the experiment. Neurological dysfunction was observed in both HIBD group and Rg1 group, showing significant difference in Longa score when compared with that in Sham group (P lt; 0.05). There was significant difference in Longa score between Rg1 group and HIBD group at 72 hours after HIBD (P lt; 0.05). Western blot showed that the protein expressions of both HIF-1α and CC3 were observed at every time point in every group. The expressions of HIF-1α protein in HIBD group and Rg1 group were significantly higher than those in Sham group at 4, 8, 24, and 72 hours (P lt; 0.05), and the expressions in Rg1 group were significantly higher than those in HIBD group (P lt; 0.05). The expressions of CC3 protein in HIBD group were significantly higher than those in Sham group at 4, 8, 24, and 72 hours (P lt; 0.05), and significant difference was found between Rg1 group and Sham group only at 4 hours (P lt; 0.05). Immunohistochemistry staining demonstrated that HIF-1α and CC3 protein mainly distributed in nucleusand cytoplasma, the results of HIF-1α and CC3 protein expression were similar to the results by Western blot. TUNEL staining showed that the positive cells were characterized by yellow or brown particle confined within nucleus. The number of apoptotic cells at every time point in HIBD group was significantly higher when compared with that in Sham group (P lt; 0.05), and the number of apoptotic cells in Rg1 group was significantly lower when compared with that in HIBD group at 8, 24, and 72 hours (P lt; 0.05). Conclusion Rg1 could inhibit Caspase 3 activation by strengthening and stabil izing HIF-1α signal pathway, and plays a role of anti-apoptosis in neonatal rats with HIBD.
OBJECTIVE: To investigate apoptosis of chondrocytes cultured in vitro and related expression of caspase-3. METHODS: Apoptosis of chondrocytes were detected by flow cytometry analysis and TUNEL staining. The expression of caspase-3 was determined by RT-PCR and Western blot, and caspase-3 protein activity was determined by ELISA. RESULTS: Apoptosis was observed in chondrocytes cultured in vitro from passage 1 to passage 4 at various degrees. The percentage of apoptosis of chondrocytes on day 7 was much higher than that on day 3 (15.7% +/- 0.3% vs 8.9% +/- 0.6%, P lt; 0.01). caspase-3 mRNA and protein expressed in chondrocytes during whole culture process. Along with the culture time extension in vitro, caspase-3 expression and protein activity up-regulated, coincident with apoptosis of chondrocyte. caspase-3 was activated and a fragment of 20 kDa was detected after 7 days of culture. CONCLUSION: caspase-3 is involved in apoptosis of chondrocytes cultured in vitro.
Objective To observe the effect of melatonin (MT) on retinal apoptosis in rats with ischemia-reperfusion injury (RIRI). Methods A total of 54 male healthy Sprague-Dawley adult rats were randomly divided into the normal control (CON) group (6 rats), RIRI group (24 rats) and MT group (24 rats). The rats of RIRI and MT group were induced using suture-occluded methods to establish RIRI model. The rats of MT group were injected with MT in the left carotid artery 30 minutes after RIRI, and RIRI group was injected with the same amount of saline. On 6, 24 hours and 3, 7 days after RIRI, the morphological changes of retina were evaluated by hematoxylin and eosin (HE) staining; the effects of MT on retinal cell apoptosis and Nrf2, HO-1 proteins were examined by immunohistochemistry staining. The correlation between active Caspase-3 and Nrf2 protein, active Caspase-3 and HO-1 protein in MT group were analyzed by linear regression analysis. Results HE staining results showed that the morphology of retinal cells was regular and retinal cells were well arranged in the CON and MT group. In the RIRI group, both the thickness of inner retinal layer and the number of retinal ganglion cells (RGC) were decreased. On 6, 24 hours and 3, 7 days after RIRI, the thickness of inner retinal layer (F=16.710, 62.303, 68.389, 57.132; P＜0.01) and RGC number (F=24.250, 11.624, 14.155, 32.442; P＜0.05) in MT group were more than those in RIRI group. Immunohistochemistry staining results showed that less active Caspase-3+ cells were observed in MT group as compared with those in RIRI group at each time points (F=49.118, 134.173, 76.225, 18.385; P＜0.01). There were more Nrf2+ (F=11.041, 31.480, 59.246, 6.740; P＜0.05) and HO-1+ cells (F=128.993, 21.606, 51.349, 8.244; P＜0.05) in MT group as compared with those in RIRI group at each time points. Linear regression analysis results showed that the difference of active Caspase-3+ cells were all linearly correlated with the Nrf2+ cells and HO-1+cells in the MT group (r2=0.810, 0.730; P＜0.01). Conclusion MT could reduce retinal cell apoptosis in RIRI rats, and its mechanism may be associated with increased Nrf2 and HO-1 expression, reduced active Caspase-3 expression.