Ascorbic acid protects male rat brain from oral potassium dichromate-induced oxdative DNA damage and apoptotic changes: the expression patterns of caspase-3, P 53, Bax, and Bcl-2 genes.

Our study designed to study the potential of potassium dichromate (K2Cr2O7) oral exposure to induce damage in male rat brain and to compare the possible protective role of vitamin C (VC) either pre and/or concurrent supply against (K2Cr2O7) induced changes. Thirty male rats were divided into five groups. First control group received distilled water (C), second received 120 mg/kg b.wt (VC), third received 25 mg/kg b.wt K2Cr2O7 (Cr), fourth group received VC together with K2Cr2O7 by the same former doses (VC + Cr), and the fifth group received the same oral doses of VC 2 weeks prior to and along with K2Cr2O7 for 6 weeks (VC + Cr pro/co treated). The obtained results revealed that K2Cr2O7 induced a significant decrease in cholinergic activity, glutathione reductase GR activity, reduced glutathione content GSH and ATP levels. Furthermore, K2Cr2O7 induced a significant increase in oxidative DNA damage indicated by 8-hydroxy 2'-deoxyguanosine (8OH2'dG) and formation of apoptotic DNA ladders, significant increase in malondialdehyde (MDA), protein carbonyl, and lactate dehydrogenase enzyme. Increased mRNA expression of pro-apoptotic genes, including caspase-3, p53, and Bax, unlike Bcl-2 expression, was decreased. K2Cr2O7 increased caspase-3 and decreased Bcl-2 immuno-labeling. VC supply noticeably ameliorates K2Cr2O7-induced changes which were more significantly in VC pro and concurrent supplement rather than VC concurrent supply only. Finally, it is concluded that K2Cr2O7 oral administration induced oxidative apoptotic changes in rat brain and confirms the usefulness of VC pre and concurrent supply for the amelioration of K2Cr2O7-induced events more significantly than VC concurrent supply only.

Effects of Yiqi Mingmu Pills on Retinal BaxmRNA Protein and Caspase-3mRNA Expression in Retinal Pigmented Rats / 世界科学技术-中医药现代化

Objective: To observe the effect of Yiqi Mingmu Pill on the expression of BaxmRNA protein and caspase-3 mRNA in retina of RCS rats. Methods: Twenty-four RCS rats were randomly divided into three groups: blank group, model group, Yiqi Mingmu Pill group. In the blank group, there were 8 RCS (rdy+/+, p+/+) rats, with 4 males and 4 females, and they received intragastric administration of normal saline. The model group consisted of 8 RCS (rdy-/, p-/-) rats, with 4 males and 4 females, and they received intragastric administration of normal saline. Yiqi Mingmu Pills groupconsisted of 8 RCS (rdy-/, p-/-) rats, with 4 males and 4 females, respectively. They received intragastric administrationof suspension of Yiqi Mingmu Pills. After intragastric administration for 30 days, the structure of retina layers wasobserved by HE staining. BaxmRNA and Caspase-3 mRNA in retinal tissues of each group were measured by RT-qPCRand Western Blot. Results: HE staining showed that the retina thickness of rats in Yiqi Mingmu Pill group wassignificantly thicker than that of the model group. The retinal pigment epithelial band was partially visible, and part of theouter nuclear layer photoreceptor sensory ciliary layer was visible. The number of photoreceptor cell nuclei was higherthan that of the model. There were many groups. The outer layer, outer core layer, and the visual cone layer were clearerand thicker than the model group. RT-qPCR showed that the relative expression of Bax mRNA and Caspase-3 mRNA inYiqi Mingmu Pills group was significantly lower than the model group, and the difference was statistically significant (P＜0.01), WB test showed that the relative expression of Bax protein and Caspase-3 protein in Yiqi Mingmu Pill group wassignificantly lower than that in the model group, and the difference was statistically significant (P＜0.01) . Conclusion: YiqiMingmu Pill has protective effect on the ultrastructure of RP retina. Yiqi Mingmu Pills can reduce the apoptosis of retinalphotoreceptor cells by inhibiting the expression of BaxmRNA and Caspase-3 mRNA on the retina and protect the visualcells.

[Effects of Acupotomy Therapy on mRNA Expressions of Bcl-2, Bax, Caspase-3 in Posterior Cervical Extensor Muscles in Cervical Spondylosis Rabbits].

OBJECTIVE: To observe the effect of acupotomy therapy on mRNA expressions of Bcl-2, Bax , Caspase-3 in posterior cervical extensor muscles in cervical spondylosis rabbits, and explore its mechanisms for apoptosis of cervical muscles. METHODS: New Zealand rabbits were randomly divided into normal, model, electroacupuncture (EA) and acupotomy groups, 6 in each group. The cervical spondylosis model was established by forced head-bowing for a long term. After model establishment, acupotomy was used at the trapezius muscle starting point and attachment site of sternocleidomastoid muscle, etc., once a week, total 3 times. EA was used at "Tianzhu" (BL 10), "Jingbailao" (EX-HN 15) and "Dazhu" (BL 11) for 3 weeks, 20 min a time, 3 times a week. Bcl-2, Bax, Caspase-3 mRNA expressions in posterior cervical extensor muscles were detected by real-time PCR. RESULTS: There was no significant difference in the expression of Bcl-2 mRNA among all the groups (P>0.05). Compared with the normal group, Bax mRNA increased in the model group (P<0.01), and that in the acupotomy group was lower than that in the model group (P<0.01). The ratio of Bcl-2/Bax mRNA in the model group decreased significantly compared with that in the normal group (P<0.01); in comparison with the model group, the ratio in the acupotomy group increased (P<0.01); and that in the acupotomy group was higher than the ratio in the EA group (P<0.05). The Caspase-3 mRNA expression in the model group increased compared with that in the normal group (P<0.05), and its expression in the acupotomy group decreased compared with those in the model and EA groups (P<0.05). CONCLUSIONS: Acupotomy therapy can regulate the mRNA expressions of Bax and Caspase-3, and retard apoptosis in posterior cervical extensor muscles, therefore the strained muscles are relieved, which may be one of its mechanisms for improving cervical spondylosis.

Effects of Acupotomy Therapy on mRNA Expressions of Bcl-2, Bax, Caspase-3 in Posterior Cervical Extensor Muscles in Cervical Spondylosis Rabbits / 针刺研究

OBJECTIVE: To observe the effect of acupotomy therapy on mRNA expressions of Bcl-2, Bax , Caspase-3 in posterior cervical extensor muscles in cervical spondylosis rabbits, and explore its mechanisms for apoptosis of cervical muscles. METHODS: New Zealand rabbits were randomly divided into normal, model, electroacupuncture (EA) and acupotomy groups, 6 in each group. The cervical spondylosis model was established by forced head-bowing for a long term. After model establishment, acupotomy was used at the trapezius muscle starting point and attachment site of sternocleidomastoid muscle, etc., once a week, total 3 times. EA was used at "Tianzhu" (BL 10), "Jingbailao" (EX-HN 15) and "Dazhu" (BL 11) for 3 weeks, 20 min a time, 3 times a week. Bcl-2, Bax, Caspase-3 mRNA expressions in posterior cervical extensor muscles were detected by real-time PCR. RESULTS: There was no significant difference in the expression of Bcl-2 mRNA among all the groups (P>0.05). Compared with the normal group, Bax mRNA increased in the model group (P<0.01), and that in the acupotomy group was lower than that in the model group (P<0.01). The ratio of Bcl-2/Bax mRNA in the model group decreased significantly compared with that in the normal group (P<0.01); in comparison with the model group, the ratio in the acupotomy group increased (P<0.01); and that in the acupotomy group was higher than the ratio in the EA group (P<0.05). The Caspase-3 mRNA expression in the model group increased compared with that in the normal group (P<0.05), and its expression in the acupotomy group decreased compared with those in the model and EA groups (P<0.05). CONCLUSIONS: Acupotomy therapy can regulate the mRNA expressions of Bax and Caspase-3, and retard apoptosis in posterior cervical extensor muscles, therefore the strained muscles are relieved, which may be one of its mechanisms for improving cervical spondylosis.

miR-155 targets Caspase-3 mRNA in activated macrophages.

To secure the functionality of activated macrophages in the innate immune response, efficient life span control is required. Recognition of bacterial lipopolysaccharides (LPS) by toll-like receptor 4 (TLR4) induces downstream signaling pathways, which merge to induce the expression of cytokine genes and anti-apoptotic genes. MicroRNAs (miRNAs) have emerged as important inflammatory response modulators, but information about their functional impact on apoptosis is scarce. To identify miRNAs differentially expressed in response to LPS, cDNA libraries from untreated and LPS-activated murine macrophages were analyzed by deep sequencing and regulated miRNA expression was verified by Northern blotting and qPCR. Employing TargetScan(TM) we identified CASPASE-3 (CASP-3) mRNA that encodes a key player in apoptosis as potential target of LPS-induced miR-155. LPS-dependent primary macrophage activation revealed TLR4-mediated enhancement of miR-155 expression and CASP-3 mRNA reduction. Endogenous CASP-3 and cleaved CASP-3 protein declined in LPS-activated macrophages. Accumulation of miR-155 and CASP-3 mRNA in miRNA-induced silencing complexes (miRISC) was demonstrated by ARGONAUTE 2 (AGO2) immunoprecipitation. Importantly, specific antagomir transfection effectively reduced mature miR-155 and resulted in significantly elevated CASP-3 mRNA levels in activated macrophages. In vitro translation assays demonstrated that the target site in the CASP-3 mRNA 3'UTR mediates miR-155-dependent Luciferase reporter mRNA destabilization. Strikingly, Annexin V staining of macrophages transfected with antagomir-155 and stimulated with LPS prior to staurosporine (SSP) treatment implied that LPS-induced miR-155 prevents apoptosis through CASP-3 mRNA down-regulation. In conclusion, we report that miR-155-mediated CASP-3 mRNA destabilization in LPS-activated RAW 264.7 macrophages suppresses apoptosis, as a prerequisite to maintain their crucial function in inflammation.

Expression of Caspase-3 mRNA in frontal cortex and hippocampus of chronic stress-induced depression rats treated by electro-acupuncture / 中华行为医学与脑科学杂志

ObjectiveTo observe the expression of Caspase-3 mRNA in prefrontal cortex and hippocampus of chronic stress-induced depression rats,and to detect the machnisms of antidepression by electro-acupuncture.MethodsSprague-Dawley rats were randomly divided into four groups:control group,model group,model +electro-acupuncture group and model + paroxetine group,12 rats in each group.Open-field test was used to observe the changes of movements,and real-time fluorescence quantitative PCR (RT-PCR) method was used to detect Caspase-3 mRNA levels in prefrontal cortex and hippocampus.Results ①Open-field test:after stress,compared with control group rats,the model group rats' crossing numbers (29 ± 7),rearing times (6 ± 2) were apparently less than those of control group( (66 ± 13),( 10 ±2) ; P＜0.05,P＞0.05).In comparison with model group,the crossing times and rearing times being increasing in degree in electro-acupuncture group( (61 ±9),( 13 ±1 ) ) and paroxetine group( (39 ± 10),(8 ± 1 ),P＜0.01,P＞0.05).② Compared with the control group,Caspase-3 mRNA in prefrontal cortex and hippocampus significantly increased in model group(P ＜0.05 ) ;and compared with model group,Caspase-3 mRNA in prefrontal cortex in electro-acupuncture group and paroxetine group significantly decreased(P ＜ 0.05 ),and both expression of Caspase-3 mRNA in hippocampus in electro-acupuncture group and expression of Caspase-3 mRNA in hippocampus in paroxetine group decreased (P ＞ 0.05 ).ConclusionChronic stress can increase the expression of Caspase-3 mRNA in prefrontal cortex and hippocampus of chronic stress-induced depression rats,while electro-acupuncture can decrease the expression of caspase-3 mRNA,which may be an important way to anti-depression by electro-acupuncture.

Effect of Bak Foong Pills on the expression of ß-amyloid in rat retina with optic nerve transection.

AIM: To investigate the effect of Bak Foong Pills (BFP) on the expression of ß-amyloid (Aß) in rats retina with optic nerve transection, and its roles and possible mechanisms in protecting optic nerve damage. METHODS: Seventy-two healthy, Sprague-Dawley, adult rats were randomly assigned to three groups: negative control group (control group), optic nerve transection group (model group) and BFP treatment group (BFP group, 100µg/mL) followed by establishing optic nerve transection model. The expression of Aß was measured at 48 hours by Western-blotting. Moreover, the expressions of Bcl-2, Bax and Caspase-3 mRNA were evaluated at 48 hours by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: There were significant differences among the control, model and BFP groups in the expression of Aß (all P<0.01). Aß expression was significantly higher in the model and BFP groups than that in the control group (P<0.01), with a more significant reduction in the BFP group than that in the model group (P<0.01). Moreover, there were also significant differences among the three groups in the expressions of Bcl-2/Bax (Bcl-2: anti-apoptotic; Bax: proapoptotic) and Caspase-3 mRNA (proapoptotic) (all P<0.01). Bcl-2/Bax ratio was significantly lower and Caspase-3 mRNA expression was significantly higher in the model and BFP groups than those in the control group (P<0.01), with a significant growing of Bcl-2/Bax and reduction of Caspase-3 in the BFP group than those in the model group (P<0.01). CONCLUSION: BFP can down-regulate Aß expression in retina and may inhibit apoptosis and protect optic nerve by enhancing Bcl-2/Bax ratio and inhibiting Caspase-3 pathway.

Effect of electroacupuncture on brain cells apoptosis and nerve function recovery after focal cerebral ischemia-reperfusion in rats / 中华行为医学与脑科学杂志

Objective To investigate the influence of acpuncture on free calcium in rat brain cells after focal cerebral ischemia reperfusion.Methods 145 adult male SD rats were randomly divided into control group,simple ischemia reperfusion group and acupuncture with ischemia reperfusion group.The middle cerebral artery occlusion/reperfusion (MCAO/R) rat model was established by the modified Longa occlusion method. ①The part of free calcium in rat brain cells,focal cevebral ischemia model of rats were made by thread locking up the blood vessel for 15 min.30 min later after reperfusion, the Baihui and Shuigou Point in Du meridian were acupunctured electrically 30 min.After 3h, 6h and 12h, the rat was killed and its brain cells were made into single cell suspension,marked by Fluo-3/AM.The fluorescence optical density was recorded by laser confocal microscopy.②The part of nerve functional reconstruction, focal cevebral ischemia model of rats were made by thread locking up the blood vessel for 12 hours.30 min later after reperfusion, the Baihui and Shuigou Point in Du meridian were acupunctured electrically 30 min.After 7 d, 14 d,30 d,60 d and 90 d, the rat was forced to detect it's strength of the dog.Results ①Free calcium in rats of acupuncture therapy group(6h:10.96±1.18;2h:20.9±4.37) was significantly less than that in control group in 6 h and 12 h after reperfusion (6 h: 16.87 ± 3.56,12 h: 34.10 ±1.06)(P<0.05).②The dog in rats of acupuncture therapy group was significantly more than that in control group in 7 d, 14 d after reperfusion (P＜ 0.05 ).No difference of the dog was detected in 30 d ,60 d and 90 d after reperfusion between the two groups.Conclusion Acupunture could decreases the concentration of free calcium and the expression of Caspase-3 mRNA in rat brain cells after focal cerebral ischemia reperfusion, and it can facilitate the recovery of nerve function.