ABSTRACT
A dual-mode nanoprobe was constructed to detect Bax messenger RNA (mRNA), consisting of gold nanotriangles (AuNTs), a Cy5-modified recognition sequence, and a thiol-modified DNA sequence. Bax mRNA is one of the key pro-apoptotic factors in the apoptosis pathway. Raman enhancement and fluorescence quenching of the signal group Cy5 were performed using AuNTs as substrates. The thiol-modified nucleic acid chain is partially complementary to the Cy5-modified nucleic acid chain to form a double strand and is linked to the AuNTs by the Au-S bond. When Bax mRNA is present, the Cy5-modified strand specifically binds to it to form a more stable duplex, making Cy5 far away from AuNTs, and SERS signal is weakened while fluorescence signal is enhanced. The nanoprobe can be used for the quantitative detection of Bax mRNA in vitro. Combined with the high sensitivity of SERS and the visualization of fluorescence, this method has good specificity and can be used for in situ imaging and dynamic monitoring of Bax mRNA during deoxynivalenol (DON) toxin-induced apoptosis of HepG2 cells. DON plays a pathogenic role mainly by inducing cell apoptosis. The results confirmed that the proposed dual-mode nanoprobe has good versatility in various human cell lines.
Subject(s)
Apoptosis , Sulfhydryl Compounds , Humans , bcl-2-Associated X Protein , RNA, Messenger , Fluorescence , Cell Line, TumorABSTRACT
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.
Subject(s)
Acupuncture Therapy , Spondylosis , Animals , Apoptosis , Caspase 3 , Proto-Oncogene Proteins c-bcl-2 , RNA, Messenger , Rabbits , bcl-2-Associated X ProteinABSTRACT
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.
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Objective To explore the effects of intraventricular administration of insulin on the expressions of Bcl-2,Bax mRNA and neuronal hippocampus apoptosis in rats after cardiopulmonary resuscitation (CPR).Methods This experiment was implemented in the animal Laboratory center of Xuanwu Hospital of Capital Medical University.Thirty male SD rats were randomly (random number)divided into three groups:control group (n=6),CPR group (n=12),insulin treated group ( n =12).CPR was performed at 6 minutes after ventricular fibrillation induced by transesophageal overdrive pacing.Resuscitation procedures lasted until restoration of spontaneous circulation (ROSC).ROSC was defined as the recovery of the supraventricular heart rates and the increase of mean arterial pressure (MAP) ＞ 60mmHg for more than 10 minutes.Ten minutes after ROSC in rats,12.5 μL ( 1 U) regular insulin was injected into the left ventricle in the insulin group,and 12.5 μL isotonic saline was injected the control and CPR groups at least 10 minutes.Real-time PCR was used to observe the expressions of Bcl-2,Bax mRNA in hippocampus CAI after reperfusion 24 h and 72 h.TUNEL staining was used to observe the neuronal apoptosis in all groups after reperfusion 7 days.Blood glucose was monitored in rats before and after CPR.Results ① The Bcl-2mRNA in insulin groups were significantly higher than those in the CPR group after 24 h and 72 h (P ＜0.01 ).The expression of Bcl-2 mRNA in 24 h insulin group were significantly higher than those in 72 h insulin group ( P ＜ 0.01 ) ; There were no significantly different in the Bax mRNA between insulin groups and the CPR and the control group after 24 h and 72 h ( P ＞ 0.05 ) ; ②After CPR 7 d,the apoptotic neurons of hippocampal CA1 area in the CPR group ( 124.75 ± 17.35 ) were significantly higher than those in the control group (5.12 ± 3.26) ( P ＜ 0.01 ) and the insulin group (92.79 ± 7.35 )(P ＜0.01 ); the apoptotic neurons in the insulin group were higher than those in the control group (P ＜0.0l ),and the differences were statistically significant.③There were no significant difference in venous blood glucose in the CPR and insulin groups (P ＞ 0.05).Conclusions Insulin may regulate Bcl-2mRNA expression in hippocampus,inhibit neuronal apoptosis and protect neurons after CPR in rats.
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Dynamic changes in mRNA expressions of liver tissue apoptosis-promoting genes Fas and Bax and apoptosis-inhibiting gene Bcl-2 of vibrio vulnificus sepsis rats were detected and the effects of antibacterial agents were examined.The rat model with Vibrio vulnificus sepsis (VV group) was established and some of the Vibrio vulnificus sepsis rats were treated with antibacterial agents (AA group).The mRNA expressions of Fas,Bax and Bcl-2 were measured by reverse transcription polymerase chain reaction (RT-PCR).As compared with normal control group (NC group),the expressions of Fas and Bax mRNA in liver tissue at all different time points in VV group were increased significantly (P<0.05),and the highest levels of Fas and Bax mRNA expressions were 6 and 12 h after the infection,respectively.At the same time,the expression of Bcl-2 mRNA in liver tissue at all different time points in VV group were decreased significantly (P<0.05),and the lowest level of Bcl-2 mRNA expression appeared 2 h after the infection.The mRNA expressions of Bcl-2 in liver tissue 9 and 12 h after the infection in AA group were increased significantly (P<0.05) compared with NC group,while the expressions of Fas and Bax mRNA were not significantly different from those of NC group.Compared with VV group,the expression of Fas mRNA in AA group was decreased (P<0.05) and Bax mRNA was decreased significantly 12 and 16 h after the infection (P<0.05),while the expressions of Bcl-2 mRNA were increased significantly 9,12 and 16 h after the infection (P<0.05).It is concluded that the mRNA expressions of liver tissue apoptosis-promoting genes Fas and Bax were increased remarkably in vibrio vulnificus sepsis rats,whereas the expression of apoptosis-inhibiting gene Bcl-2 mRNA was decreased obviously in sepsis rats in early stage.The treatment with cefoperazone sodium and levofloxacin lactate could inhibit the expression of Fas mR.NA and Bax mRNA and enhance the expression of Bcl-2 mRNA at the same time.
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OBJECTIVE:To observe the protective effect of naloxone on ischemical reperfusion injury in rat brain. METHODS: 36 Wistar rats were randomly divided into 3 groups(n=12): normal control group,model control group (normal saline) and naloxone group (naloxone 0.4 mg?kg-1). The model control group and the naloxone group were established into ischemical reperfusion injury model by a drain method of carotid artery. The naloxone was administered at the beginning of ischemia and reperfusion,respectively. At 120 min of reperfusion,the neuron morphous was observed under light microscope and electronic microscope,respectively,the width of the bright bands of the area surrounding neuron and blood vessels and the average density and gray scale value of Bax mRNA positive cells were detected. RESULTS: In naloxone group compared with model control group,the cellular morphous of the neurons was more integrated,the bright bands of the area surrounding neuron and blood vessels were narrower,the average density was lower while the gray scale value was higher for Bax mRNA positive cells and the expression of which was attenuated. CONCLUSION: The protective effect of naloxone on cerebral ischemia reperfusion injury in rat might be associated to the down-regulation of Bax mRNA expression of apoptotic cells.
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Objective: To investigate the mechanism of butyrate,which is the by-products of diet fiber fermented in colon and induce the apoptosis of human colon cancer cell. Methods: The subculture of human colon cancer cell line SW1116 was cultured in medium with different concentration of butyrate(0、2 、3 、4、7 and 10 mmol/L).The mRNA and proteins of bcl-2 or bax in cell lines were detected respectively by RT-PCR and Western Blotting while the concentration of p53 protein and the activity of caspase-3 was detected by flow cytometer and fluorescent quantitative assay respectively after 6、24、48 and 72h cultivation.Results: There was no expression of bcl-2 mRNA and its protein in SW1116 cell during the study periods but the expression of bax mRNA and its protein,the p53 protein concentration and the caspase-3 activity increased gradually when the butyrate concentration was increased and the cultivation time was prolonged. Conclusion: By up-regulating the expression of bax mRNA and its protein,butyrate induce the apoptosis of colon cancer cell on a p53 dependent way.
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AIM: To observe the protective effect of naoyikang-containing serum on cultured hippocampus neuron injury induced by D-galactose(D-gal).METHODS: Naoyikang-containing serum was prepared in rats administered with aqueous extract of naoyikang(6.84 g?kg-1?d-1).MTT,MAO-B and ATPase assay were used to measure the viability of hippocampus neurons.RESULTS: D-gal at concentration of 100 ?mol/L caused significant decrease in the viability of hippocampus neurons 24 h after the treatment.Naoyikang-containing serum increased the viability,ATPase activity and the expression of bcl-xl mRNA,decreased the MAO-B activity and the expression of bax mRNA in D-gal injured hippocampus neurons.CONCLUSION: Naoyikang-containing serum prevents hippocampus neurons from D-gal induced cytotoxicity.
