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Heroin activates ATF3 and CytC via c-Jun N-terminal kinase pathways to mediate neuronal apoptosis.

Pu H, Wang X, Su L, Ma C, Zhang Y, Zhang L, Chen X, Li X, Wang H, Liu X, Zhang J - Med Sci Monit Basic Res (2015)

Bottom Line: RESULTS We found that heroin induces the apoptosis of primary cultured cerebellar granule cells (CGCS) and that the c-Jun N-terminal kinase (JNK) pathway was activated under heroin treatment and stimulated obvious increases in the levels of C-jun, Cytc, and ATF3mRNA.The results suggested that SP600125 of JNK/C-jun can inhibit heroin-induced apoptosis of neurons.CONCLUSIONS The present study analyzes our understanding of the critical role of the JNK pathway in the process of neuronal apoptosis induced by heroin, and suggests a new and effective strategy to treat SLE.

View Article: PubMed Central - PubMed

Affiliation: Department of Science and Research Education Center, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Sinciang, China (mainland).

ABSTRACT
BACKGROUND Drug abuse and addiction has become a major public health problem that impacts all societies. The use of heroin may cause spongiform leukoencephalopathy (SLE). MATERIAL AND METHODS Cerebellar granule cells were derived from 7-day-old Sprague-Dawley rat pups. Neurons were dissociated from freshly dissected cerebella by mechanical disruption in the presence of 0.125% trypsin and DNaseI and then seeded at a density of 4×10^6 cells/ml in Dulbecco's modified Eagle's medium/nutrient mixture F-12 ham's containing 10% fetal bovine serum and Arc-C(sigma) at concentrations to inhibit glial cell growth inoculated into 6-well plates and a small dish. RESULTS We found that heroin induces the apoptosis of primary cultured cerebellar granule cells (CGCS) and that the c-Jun N-terminal kinase (JNK) pathway was activated under heroin treatment and stimulated obvious increases in the levels of C-jun, Cytc, and ATF3mRNA. CYTC and ATF3 were identified as candidate targets of the JNK/c-Jun pathway in this process because the specificity inhibitors SP600125 of JNK/C-jun pathways reduced the levels of C-jun, Cytc, and ATF3mRNA. The results suggested that SP600125 of JNK/C-jun can inhibit heroin-induced apoptosis of neurons. CONCLUSIONS The present study analyzes our understanding of the critical role of the JNK pathway in the process of neuronal apoptosis induced by heroin, and suggests a new and effective strategy to treat SLE.

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JNK/c-Jun pathway regulates Cytc and ATF3 induction in heroin-induced neuronal apoptosis. (A) DIV7 CGNs were placed in media with or without different dosage of heroin for 24 h (a=normal medium, b=10 ug/ml, c=40 ug/ml, d=80 ug/ml, e=120 ug/ml), then neurons were assessed by PCR using the indicated the adopt of p-c-jun, cytc, ATF3 and β-actin primer (A1) and RT-PCR using the p-c-jun, cytc, ATF3 and β-actin were quantified (A2–A4). (B) DIV7 CGNs were placed in media with or without different dosage of heroin for 24 h (a=normal medium, b=10 ug/ml, c=40 ug/ml, d=80 ug/ml, e=120 ug/ml), then neurons were assessed by Western Blotting using the indicated the adopt of p-c-jun, cytc, ATF3 and β-tubulin protein (B2–B4). (C) DIV7 CGNs were transferred to media containing 120 ug/ml heroin in the absence or presence of 10 um SP600125.After 24 h, neurons were assessed by PCR using the indicated Semi-quantitative (C1) and RT-PCR using the p-c-jun, cytc and β-actin were quantified (C2–C4). (D) DIV7 CGNs were transferred to media containing 120 ug/ml heroin in the absence or presence of 10 um SP600125. After 24 h, neurons were assessed by Western Blotting using the p-c-jun, cytc and β-tubulin protein were quantified (D2–D4).* p<0.05, **p<0.01.
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f5-medscimonitbasicres-21-53: JNK/c-Jun pathway regulates Cytc and ATF3 induction in heroin-induced neuronal apoptosis. (A) DIV7 CGNs were placed in media with or without different dosage of heroin for 24 h (a=normal medium, b=10 ug/ml, c=40 ug/ml, d=80 ug/ml, e=120 ug/ml), then neurons were assessed by PCR using the indicated the adopt of p-c-jun, cytc, ATF3 and β-actin primer (A1) and RT-PCR using the p-c-jun, cytc, ATF3 and β-actin were quantified (A2–A4). (B) DIV7 CGNs were placed in media with or without different dosage of heroin for 24 h (a=normal medium, b=10 ug/ml, c=40 ug/ml, d=80 ug/ml, e=120 ug/ml), then neurons were assessed by Western Blotting using the indicated the adopt of p-c-jun, cytc, ATF3 and β-tubulin protein (B2–B4). (C) DIV7 CGNs were transferred to media containing 120 ug/ml heroin in the absence or presence of 10 um SP600125.After 24 h, neurons were assessed by PCR using the indicated Semi-quantitative (C1) and RT-PCR using the p-c-jun, cytc and β-actin were quantified (C2–C4). (D) DIV7 CGNs were transferred to media containing 120 ug/ml heroin in the absence or presence of 10 um SP600125. After 24 h, neurons were assessed by Western Blotting using the p-c-jun, cytc and β-tubulin protein were quantified (D2–D4).* p<0.05, **p<0.01.

Mentions: We wondered whether ATF3 and Cytc were closely related with heroin-induced apoptosis and how C-jun acted as a target gene in the JNK pathway. Therefore, we studied the mechanism by which Cytc and ATF3 are regulated under heroin intoxication through JNK pathway. We started with observing the expression level of p-c-jun, cytc, and ATF3 mRNA in different concentrations of heroin. As shown in Figure 5A, we found that expression levels of p-c-jun, cytc and ATF3 mRNA began to up-regulate with increasing concentrations of heroin medium (10 ug/ml, 40 ug/ml, 80 ug/ml, 100 ug/ml, and 120 ug/ml). In this process, the β-actin gene acted as an internal reference. By reverse transcription polymerase chain reaction (RT-PCR) detection, we compared the results with the control group, and found that the expression level of P-c-jun mRNA reached a peak at a heroin concentration of 120 ug/ml (Figure 5A2). The expression level of Cytc mRNA was far higher compared to the control group without heroin (Figure 5A3), and the expression level of ATF3 mRNA was also increased dramatically (Figure 5A4). The same result applies to the effect of the Western blotting. P-c-jun, cytc and ATF3 protein expression levels began to up-regulate with increasing doses of heroin (10 ug/ml, 40 ug/ml, 80 ug/ml, 100 ug/ml and 120 ug/ml) (Figure 5B). It was shown that P-c-jun, Cytc and ATF3 factors involved in the process of neuronal apoptosis by heroin when P-c-jun, cytc and ATF3 mRNA and protein level were shown dose-denpendent with heroin. Then, we observed the expression level of p-c-jun, cytc and ATF3 mRNA in H group (120 ug/ml heroin), H+S group (120 ug/ml heroin +10 uM SP600125) and NM group(control group) with medium. As shown in Figure 5C, we found that expression levels of p-c-jun, cytc and ATF3 mRNA reduced obviously in H+S group through ordinary polymerase chain reaction(PCR) detecting (Figure 5C1). By reverse transcription polymerase chain reaction (RT-PCR) detection, The results compared with the H group, expression level of P-c-jun mRNA was reaching a lower peak in 120 ug/ml heroin and 10 uM SP600125 (H+S group) (Figure 5C2), expression level of Cytc mRNA was decreased compared to H group (Figure 5C3), expression level of ATF3 mRNA was also decreased dramatically (Figure 5C4). Meanwhile, P-c-jun, cytc and ATF3 protein expression levels were similarity to their mRNA expression in three groups (NM group, H group and H+S group) by western blotting detected (Figure 5D1). We also found the expression level of P-c-jun, cytc and ATF3 protein were decreased obviously in H+S group compared with H group (*P<0.05) (Figure 5D2–D4]. With applying of JNK pathway inhibitor SP600125, P-c-jun, Cytc and ATF3 mRNA and protein expression levels had downward trend compared the single factor of heroin (120 ug/ml) which intervened in CGNs (Figure 5C, D1). The expression levels of P-c-jun, Cytc and ATF3 mRNA and proteins were significantly decreased which also indicated Cytc and ATF3 played a vital role in promoting apoptosis as C-jun candidate genes through JNK pathway. All these datas indicated that P-c-jun, Cytc and ATF3 factors were involved in the process of heroin-induced CGN apoptosis.


Heroin activates ATF3 and CytC via c-Jun N-terminal kinase pathways to mediate neuronal apoptosis.

Pu H, Wang X, Su L, Ma C, Zhang Y, Zhang L, Chen X, Li X, Wang H, Liu X, Zhang J - Med Sci Monit Basic Res (2015)

JNK/c-Jun pathway regulates Cytc and ATF3 induction in heroin-induced neuronal apoptosis. (A) DIV7 CGNs were placed in media with or without different dosage of heroin for 24 h (a=normal medium, b=10 ug/ml, c=40 ug/ml, d=80 ug/ml, e=120 ug/ml), then neurons were assessed by PCR using the indicated the adopt of p-c-jun, cytc, ATF3 and β-actin primer (A1) and RT-PCR using the p-c-jun, cytc, ATF3 and β-actin were quantified (A2–A4). (B) DIV7 CGNs were placed in media with or without different dosage of heroin for 24 h (a=normal medium, b=10 ug/ml, c=40 ug/ml, d=80 ug/ml, e=120 ug/ml), then neurons were assessed by Western Blotting using the indicated the adopt of p-c-jun, cytc, ATF3 and β-tubulin protein (B2–B4). (C) DIV7 CGNs were transferred to media containing 120 ug/ml heroin in the absence or presence of 10 um SP600125.After 24 h, neurons were assessed by PCR using the indicated Semi-quantitative (C1) and RT-PCR using the p-c-jun, cytc and β-actin were quantified (C2–C4). (D) DIV7 CGNs were transferred to media containing 120 ug/ml heroin in the absence or presence of 10 um SP600125. After 24 h, neurons were assessed by Western Blotting using the p-c-jun, cytc and β-tubulin protein were quantified (D2–D4).* p<0.05, **p<0.01.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4400970&req=5

f5-medscimonitbasicres-21-53: JNK/c-Jun pathway regulates Cytc and ATF3 induction in heroin-induced neuronal apoptosis. (A) DIV7 CGNs were placed in media with or without different dosage of heroin for 24 h (a=normal medium, b=10 ug/ml, c=40 ug/ml, d=80 ug/ml, e=120 ug/ml), then neurons were assessed by PCR using the indicated the adopt of p-c-jun, cytc, ATF3 and β-actin primer (A1) and RT-PCR using the p-c-jun, cytc, ATF3 and β-actin were quantified (A2–A4). (B) DIV7 CGNs were placed in media with or without different dosage of heroin for 24 h (a=normal medium, b=10 ug/ml, c=40 ug/ml, d=80 ug/ml, e=120 ug/ml), then neurons were assessed by Western Blotting using the indicated the adopt of p-c-jun, cytc, ATF3 and β-tubulin protein (B2–B4). (C) DIV7 CGNs were transferred to media containing 120 ug/ml heroin in the absence or presence of 10 um SP600125.After 24 h, neurons were assessed by PCR using the indicated Semi-quantitative (C1) and RT-PCR using the p-c-jun, cytc and β-actin were quantified (C2–C4). (D) DIV7 CGNs were transferred to media containing 120 ug/ml heroin in the absence or presence of 10 um SP600125. After 24 h, neurons were assessed by Western Blotting using the p-c-jun, cytc and β-tubulin protein were quantified (D2–D4).* p<0.05, **p<0.01.
Mentions: We wondered whether ATF3 and Cytc were closely related with heroin-induced apoptosis and how C-jun acted as a target gene in the JNK pathway. Therefore, we studied the mechanism by which Cytc and ATF3 are regulated under heroin intoxication through JNK pathway. We started with observing the expression level of p-c-jun, cytc, and ATF3 mRNA in different concentrations of heroin. As shown in Figure 5A, we found that expression levels of p-c-jun, cytc and ATF3 mRNA began to up-regulate with increasing concentrations of heroin medium (10 ug/ml, 40 ug/ml, 80 ug/ml, 100 ug/ml, and 120 ug/ml). In this process, the β-actin gene acted as an internal reference. By reverse transcription polymerase chain reaction (RT-PCR) detection, we compared the results with the control group, and found that the expression level of P-c-jun mRNA reached a peak at a heroin concentration of 120 ug/ml (Figure 5A2). The expression level of Cytc mRNA was far higher compared to the control group without heroin (Figure 5A3), and the expression level of ATF3 mRNA was also increased dramatically (Figure 5A4). The same result applies to the effect of the Western blotting. P-c-jun, cytc and ATF3 protein expression levels began to up-regulate with increasing doses of heroin (10 ug/ml, 40 ug/ml, 80 ug/ml, 100 ug/ml and 120 ug/ml) (Figure 5B). It was shown that P-c-jun, Cytc and ATF3 factors involved in the process of neuronal apoptosis by heroin when P-c-jun, cytc and ATF3 mRNA and protein level were shown dose-denpendent with heroin. Then, we observed the expression level of p-c-jun, cytc and ATF3 mRNA in H group (120 ug/ml heroin), H+S group (120 ug/ml heroin +10 uM SP600125) and NM group(control group) with medium. As shown in Figure 5C, we found that expression levels of p-c-jun, cytc and ATF3 mRNA reduced obviously in H+S group through ordinary polymerase chain reaction(PCR) detecting (Figure 5C1). By reverse transcription polymerase chain reaction (RT-PCR) detection, The results compared with the H group, expression level of P-c-jun mRNA was reaching a lower peak in 120 ug/ml heroin and 10 uM SP600125 (H+S group) (Figure 5C2), expression level of Cytc mRNA was decreased compared to H group (Figure 5C3), expression level of ATF3 mRNA was also decreased dramatically (Figure 5C4). Meanwhile, P-c-jun, cytc and ATF3 protein expression levels were similarity to their mRNA expression in three groups (NM group, H group and H+S group) by western blotting detected (Figure 5D1). We also found the expression level of P-c-jun, cytc and ATF3 protein were decreased obviously in H+S group compared with H group (*P<0.05) (Figure 5D2–D4]. With applying of JNK pathway inhibitor SP600125, P-c-jun, Cytc and ATF3 mRNA and protein expression levels had downward trend compared the single factor of heroin (120 ug/ml) which intervened in CGNs (Figure 5C, D1). The expression levels of P-c-jun, Cytc and ATF3 mRNA and proteins were significantly decreased which also indicated Cytc and ATF3 played a vital role in promoting apoptosis as C-jun candidate genes through JNK pathway. All these datas indicated that P-c-jun, Cytc and ATF3 factors were involved in the process of heroin-induced CGN apoptosis.

Bottom Line: RESULTS We found that heroin induces the apoptosis of primary cultured cerebellar granule cells (CGCS) and that the c-Jun N-terminal kinase (JNK) pathway was activated under heroin treatment and stimulated obvious increases in the levels of C-jun, Cytc, and ATF3mRNA.The results suggested that SP600125 of JNK/C-jun can inhibit heroin-induced apoptosis of neurons.CONCLUSIONS The present study analyzes our understanding of the critical role of the JNK pathway in the process of neuronal apoptosis induced by heroin, and suggests a new and effective strategy to treat SLE.

View Article: PubMed Central - PubMed

Affiliation: Department of Science and Research Education Center, First Affiliated Hospital of Xinjiang Medical University, Urumqi, Sinciang, China (mainland).

ABSTRACT
BACKGROUND Drug abuse and addiction has become a major public health problem that impacts all societies. The use of heroin may cause spongiform leukoencephalopathy (SLE). MATERIAL AND METHODS Cerebellar granule cells were derived from 7-day-old Sprague-Dawley rat pups. Neurons were dissociated from freshly dissected cerebella by mechanical disruption in the presence of 0.125% trypsin and DNaseI and then seeded at a density of 4×10^6 cells/ml in Dulbecco's modified Eagle's medium/nutrient mixture F-12 ham's containing 10% fetal bovine serum and Arc-C(sigma) at concentrations to inhibit glial cell growth inoculated into 6-well plates and a small dish. RESULTS We found that heroin induces the apoptosis of primary cultured cerebellar granule cells (CGCS) and that the c-Jun N-terminal kinase (JNK) pathway was activated under heroin treatment and stimulated obvious increases in the levels of C-jun, Cytc, and ATF3mRNA. CYTC and ATF3 were identified as candidate targets of the JNK/c-Jun pathway in this process because the specificity inhibitors SP600125 of JNK/C-jun pathways reduced the levels of C-jun, Cytc, and ATF3mRNA. The results suggested that SP600125 of JNK/C-jun can inhibit heroin-induced apoptosis of neurons. CONCLUSIONS The present study analyzes our understanding of the critical role of the JNK pathway in the process of neuronal apoptosis induced by heroin, and suggests a new and effective strategy to treat SLE.

Show MeSH
Related in: MedlinePlus