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ObjectiveTo investigate the mechanism of salvianolic acid F （Sal F） in repairing the high glucose-induced injury in human kidney-2 （HK-2） cells via the B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）/cysteinyl aspartate-specific proteinase 3 （Caspase-3）/gasdermin-E （GSDME） pathway. MethodThe cell counting kit-8 （CCK-8） was used to measure the relative viability of HK-2 cells exposed to high glucose and different concentrations （2.5， 5， 10， 20 μmol·L-1） of Sal F and the relative viability of HK-2 cells treated with Sal F for different time periods. The levels of lactate dehydrogenase （LDH） and interleukin-1β （IL-1β） in the supernatant of the cell culture were measured by the LDH assay kit and enzyme-linked immunosorbent assay （ELISA） kit， respectively. Flow cytometry combined with Annexin V-FITC/propidium iodide （PI） and Hoechst 33342/PI staining was employed to reveal the proportion of PI-positive HK-2 cells exposed to high glucose. Western blotting was employed to determine the protein levels of Bax， Bcl-2， cytochrome C， cysteinyl aspartate-specific proteinase （Caspase）-9， Caspase-3， and GSDME in the HK-2 cells exposed to high glucose and treated with Sal F. The 2，7-dichlorodihydrofluorescein diacetate fluorescence probe （DCFH-DA） and mitochondrial membrane potential assay kit （JC-1） were used to determine the production of reactive oxygen species （ROS） and the mitochondrial membrane potential in the HK-2 cells exposed to high glucose and treated with Sal F. ResultCompared with the blank group， the model group showed decreased cell viability （P<0.01）， elevated levels LDH and IL-1β， increased proportion of PI-positive cells （P<0.01）， up-regulated protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME （P<0.01）， down-regulated protein level of Bcl-2 （P<0.01）， decreased mitochondrial membrane potential， and excessive ROS accumulation. Compared with the model group， Sal F repaired the high glucose-induced injury in HK-2 cells （P<0.05）， lowered the levels of LDH and IL-1β （P<0.05， P<0.01）， and decreased the proportion of PI-positive cells （P<0.01）. In addition， Sal F down-regulated the protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME and up-regulated the protein level of Bcl-2 （P<0.05， P<0.01）， increased the mitochondrial membrane potential， and decreased the accumulation of ROS in HK-2 cells. ConclusionSal F can reduce the production of ROS， restore the balance of mitochondrial membrane potential， and inhibit pyroptosis via the Bax/Caspase-3/GSDME signaling pathway to repair the high glucose-induced injury in HK-2 cells.
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The study is designed to evaluate the protective effect of xanthan gum (XG) injection on cartilage injury in the rabbit osteoarthritis (OA) model induced by anterior cruciate ligament transection (ACLT), and to explore the effect of XG on the expression of caspase-3 and Bax protein in OA cartilage. Sixty male New Zealand white rabbits were randomly divided into 6 groups (n=10) according to random number table method, and one group was selected randomly as the normal control group (control) while the other 5 groups of right knee were used to establish the OA model with ACLT, which were then divided into model group (model), XG-0.6 mg·kg-1, XG-1.2 mg·kg-1, XG-2.4 mg·kg-1 treatment group and sodium hyaluronate (SH-1.2 mg·kg-1) treatment group according to drug intervention. The knee joint temperature and knee joint width of each group were measured in the course of treatment. After treatment, the macroscopic morphology of rabbit joints in each group was observed. The pathological morphology of articular cartilage of rabbits in each group was observed using HE staining. The expression of Bax and cleaved caspase-3 in the cartilage of rabbits were detected by Western blot. The result shows that XG inhibited the increase in knee joint temperature and knee width caused by OA in a dose-dependent manner. XG improved the morphological abnormalities and tissue injuries of the femoral condyle and tibial plateau caused by OA. Western blot result shows that, compared with the control group, the levels of Bax and cleaved caspase-3 in knee cartilage cells of model group and XG-0.6 mg·kg-1 group were significantly increased (P-1 group (P>0.05). These two groups are significantly higher than those of XG-1.2 mg·kg-1 and XG-2.4 mg·kg-1 (P-1 and XG-1.2 mg·kg-1 group (P>0.05). The level of Bax in knee cartilage in XG-2.4 mg·kg-1 group was lower than that of XG-1.2 mg·kg-1 group (P<0.05). In conclusion, XG effectively protected cartilage damage in OA, and inhibited the expression of Bax and caspase-3 protein in OA cartilage.
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Objective To investigate the effects of self-designed Chinese medicine decoction on the expression of c-myc, caspase-3 in the rats with precancerous lesion of chronic atrophic gastritis. Methods The 120 Wistar rats were randomly divided into the normal group, the model group, the Weifuchun group and the high-, medium-and low-dose decoction groups, with 20 rats each group. Except the normal group, the rats in the other groups were prepared for chronic atrophic gastritis precancerous lesion model. After establishing the models successfully, the rats of the high-, medium-and low-dose groups were given decoction via intragastric administration which contained crude drug 2.088, 1.044, 0.522 g/ml respectively, and the Weifuchun group was given drug liquid of Weifuchun with the concentration of 0.076 g/ml. The normal group and the model group were given the equal volume of normal saline. Medicines were given once a day for 12 weeks. Then the expression of c-myc and caspase-3 in the gastric mucosa of rats were detected by using immunohistochemical staining. Results Compared to the model group, the expression of c-myc in the high-, medium-dose decoction groups and the Weifuchun group (30.25%± 3.56 %, 45.37% ± 4.81%, 38.79%± 4.02% vs. 63.45% ± 7.08%) significantly decreased (P<0.01 or P<0.05);and the expression of caspase-3 (36.85%± 5.02%, 31.24%± 4.55%, 31.57%± 4.62%vs. 23.43%± 2.95%) significantly increased (P<0.01 or P<0.05). Conclusions Self-designed Chinese medicine decoction could inhibit the expression of c-myc, and activate caspase-3 expression,which may be one of the mechanisms of treating the disease.
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Objective To study the expressions of cysteinyl aspartate specific proteinase 3 ( Caspase 3 ) and proliferating cell nuclear antigen ( PCNA ) in intestinal tissue of neonatal rats with necrotizing enterocolitis ( NEC ) , and the protective effect of glutamine ( Gln ) on NEC. MethodsThirty-six neonatal Sprague-Dawley ( SD) rats were randomly assigned into 3 groups at 48 h after birth (12 in each group). The control group were fed with milk replacer. The NEC group were fed with milk replacer and experiencing cold exposure after hypoxic-reoxygenation twice a day for 3 days, The Gln+NEC group were fed with milk replacer plus Gln and experiencing cold exposure after hypoxic-reoxygenation twice a day for 3 days. All the rats were sacrificed and intestinal tissues obtained at day 3 of the establishment of model. The histological changes of ileal tissues were studied using hematoxy lin-eosin ( HE ) staining. The expressions of Caspase 3 and PCNA were detected using immunohistochemical(IHC)method.Results Caspase3expressioninNECgroup(77.3±8.6)℅was significantly higher than the control group (18. 9 ± 3. 4)℅ and Gln+NEC group (50. 3 ± 6. 2)℅ ( P<0. 05). Also, Caspase 3 in Gln+NEC group was significantly higher than the control group (P<0. 05). PCNA expression in the NEC group ( 15. 0 ± 1. 9 )℅ was significantly lower than the control group (34. 2 ± 5. 8)℅ and the Gln +NEC group ( 24. 0 ± 3. 9 )℅ ( P <0. 05 ) . PCNA expression in the Gln+NEC group was significantly lower than the control group ( P<0. 05). The pathological score of the intestinal tissues was significantly correlated with Caspase 3 expression ( r = 0. 769, P = 0. 005 ), Caspase3/PCNA ratio (r=0. 835,P=0. 002) and PCNA expression (r= -0. 698, P=0. 014) in the NECgroup.Conclusions Up regulation of Cas pase3 and down regulation of PCNA might be correlated with the process of NEC. Gln might be effective in prevention and healing of NEC by inhibiting apoptosis and promoting cell proliferation.
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Objective To investigate the effect of hyperoxia on the expressions of cysteinyl aspartate specific proteinase -3(Caspase -3)and proliferating cell nuclear antigen (PCNA)in bone marrow mesenchymal stem cells (BMSCs).Methods Primary BMSCs from SD rats were cultured in vitro,BMSCs grew to 70% -80% degrees of fu-sion and then were randomly divided into room air group and hyperoxia exposure group.Each group was divided into 5 sub -groups,and cultured for 0,3,6,1 2,and 24 h respectively.Morphology investigation with inverted phase contrast microscope was adopted.The expressions of Caspase -3 and PCNA protein levels were detected by Western blot. Results (1 )As time wenty by,compared with room air group,the gap between cells increased,some of the cells be-came circular,and cell detachment and floating appeared in the hyperoxia -exposure group (>1 2 h)compared with room air group.(2)As time went on,compared with the room air group,the expression levels of Caspase -3 protein in the hyperoxia -exposure group were increased,and the difference was significant after 6 hours of culture (0.27 ± 0.04 vs 0.1 4 ±0.02,t =5.03,P =0.007).(3)Compared with room air group,the PCNA levels of the hyperoxiaexpo-sure group(6 h)decreased,and the difference in PCNA protein expression levels was significant after 6 hours of culture (0.27 ±0.04 vs 0.38 ±0.04,t =3.37,P =0.028).Conclusions Hyperoxia exposure increases Caspase -3 expres-sion levels and decreases PCNA expression,which may affect the proliferation and apoptosis of BMSCs.
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Objective To investigate the expression of apoptotic protease cysteinyl aspartate specific proteinase (caspase-3) in cortex neurons of rats with sepsis.Methods Models of rats with sepsis were established by the cecal ligation and puncture (CLP).Totally 70 cases of 30-day-old male Wistar rats were randomly divided into CLP group (n =50) and control group (n =20).In CLP group,CLP was performed in the rats.Neurobehavioral score was measured in 5 rats at 6,12,24 and 48 h after CLP surgery,respectively.Then,they were killed and their brains were removed.The immunohistochemical staining and Western blot were used to detect the apoptotic protein caspase-3 expression in cortex of rats.Control group did not undergo CLP,and the other treatment was the same as CLP group.Results Neurobehavioral scores at 12,24 and 48 h after CLP surgery were significantly lower than that in the control group(t =3.651,3.773,7.155,all P ＜ 0.05),and the scores were gradually decreased,overall situation of rats was getting worse along with the time.Caspase-3 protein expressed only in trace amounts in rat cerebral cortex in the control group by immunofluorescence analysis,however,its expression was significantly increased at 12 h after CLP surgery.Western blot test showed that caspase-3 protein expression in rat cerebral cortex at 6,12 and 24 h after CLP surgery was significantly higher than those in control group (all P ＜ 0.05).Its expression began to increase at 6 h after CLP surgery,and reached the peak at 12 h,then decreased at 48 h.Conclusion The neurobehavioral scores decreases and the expression of apeptosis protease caspase-3 increases in cortex of rats with sepsis brain injury.