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Protective effect of intratracheally administered antiflammin-1 on bleomycin-induced lung fibrosis
Abstracts / Cell Biology International 32 (2008) S1eS67 Tissue damage, inflammation or injury of the nervous system may result in chronic neuropathic pain characterized by sensitivity to painful stimuli (hyperalgesia), the perception of innocuous stimuli as painful (allodynia) and spontaneous pain. The P2X3 receptors play a crucial role in facilitating pain transmission at primary sensory afferent. Ferulic acid (FA) is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury. The present research investigated the effects of FA on the primary sensory afferent transmission induced by P2X3 receptor in neuropathic pain states. Chronic constriction injury (CCI) model was adopted. Sprague-Dawley male rats were randomly divided into normal saline (NS) group (I), sham group (II), CCI group (III), CCI + FA group (IV), and FA group (V). Mechanical withdrawal threshold and thermal withdrawal latency were measured and P2X3 protein in L4/L5 dorsal root ganglion (DRG) was detected. The mechanical withdrawal threshold and thermal withdrawal latency in group III were lower than group I, II, IV and V (p<0.05), while the expression of P2X3 protein in L4/L5 DRG of group III was higher than group I, II, IV and V (p<0.01). The mechanical withdrawal threshold, thermal withdrawal latency and P2X3 protein of L4/L5 DRG in group IV showed no significant difference compared with group I, group II or group V (p>0.05). The amplitude of the currents in group III (CCI) was much larger than those obtained in other groups after application of the same concentration ATP (P<0.01). a, b-MeATP (a selective agonist of P2X3 receptor) activated current in DRG neurons of CCI rats was more evident than those obtained in other group rats (P<0.01). The results showed that FA could inhibit the primary sensory afferent transmission of neuropathic pain induced by P2X3 receptor. This work was supported by the National Natural Science Foundation of China (No. 30260030) and the National Natural Science Foundation of Jiangxi Province (No. 0640062).
MICRORNA-375 PROMOTES 3T3-L1 PROADIPOCYTE DIFFERENTIATION Hong Yan Ling 1,2, Xiao Yong Lei 1, Zhi Ping Gao 1, Wei Dong Yin 1, He Sheng Ou 1, Duan Fang Liao* 1 1 Division of Pharmacoproteomics, Institute of Pharmacy and Pharmacology, University of South China, Hengyang, China 2 Department of Pharmacology, School of Pharmaceutical Science, Central South University, Changsha, China MicroRNAs (miRNAs) represent a class of short (~22 nt), noncoding regulatory RNAs which are involved in multiple biological processes such as energy homeostasis, sugar and lipid metabolism, and cell differentiation by negatively regulate gene expression at the post-transcriptional level. To investigate the effect of microRNA-375 on differentiation of 3T3-L1 preadipocyte (3T3-L1 cell), 3T3-L1 cells were cultured and transfected with microRNA-375 plasmids, and were induced to differentiation using with 0.5 mmol/L 3-isobutyl1-methyxanthine, 1 mg/L insulin, and 1mmol/ L dexamethasone. Oil red O staining was performed and marker genes of preadipocyte differentiation were detected by using real-time PCR. Results showed that the microRNA375-transfected 3T3-L1 cells contained many more small lipid droplets than did non-transfected 3T3-L1 cells before induced. Following induction, differentiation in the microRNA-375-transfected 3T3-L1 cells was dramatically promoted. The expression of adipocyte differentiation marker genes such as lipoprotein lipase (LPL), adipocyte fatty acid-binding protein (aP2) and peroxisome proliferator-activated receptor gamma (PPARgamma) were upregulated in the microRNA-375-transfected cells, whereas expression of preadipocyte factor-1 (Pref-1), an inhibitor of preadipocyte differentiation, was downregulated. These results suggest that microRNA-375 could promote 3T3-L1 preadipocyte differentiation, indicating microRNA-375 may be of potential use in the treatment of obesity and obesity-induced insulin resistance.
APPLICATION OF A BACTERIAL TWO-HYBRID SYSTEM FOR THE ANALYSIS OF BRS-3 INTERACTED PROTEINS Hui Jun Liu, Yu Rong Tan, Yang Xiang, Xiao Yan Zhou, Fei Qu, Chi Liu, Xiao Ling Zhu, Xiao Qun Qin* Department of Physiology, Xiangya School of Medicine, Central South University, Changsha, 410078, China
S59
Proteineprotein interactions play an important role in virtually all cellular processes including translation and modification of proteins, living things respondent reaction, the signal transduction and a lot of metabolic process. The bacterial two-hybrid (BTH) system has become a widely used tool for determining such interactions in vivo. Since there is very little known about the biological function, we have screened the interacted proteins of BRS-3 by using BTH technology. The pBT/BRS-3 bait plasmid was constructed and verified by double digestion and sequencing. Western blot analysis confirmed that the molecular weight of recombinant fusion protein was about 70 kDa, which was unanimous with a predicted molecular weight. Since the bait protein alone cannot activate the transcription of reporter gene, it can be used for screening of fetal brain library. Interactions were determined to be positive as measured by growth on "Selective Screening Medium" and validated by growth on "Dual Selective Screening Medium". To identify the proteins encoded by the target DNAs, the nucleotide of the target DNA was sequencing and compared to nucleotide databases to identify related proteins. All together 9 kinds of proteins and 4 kinds of new genes were identified. The findings of the BTH screen were confirmed by the glutathione S-transferase (GST)-pull down assay. These results showed that BRS-3 could interact with GRP, CNTF, SERPINB5, SERPINE1, HPN, PTK6, CSNK2A1, PRKCB1 and BIRC5. These proteins have a wide range of functions including cell growth, differentiation, anti - apoptosis, cytoskeleton construction, and the tyrosine kinase activities.
PROTECTIVE EFFECT OF INTRATRACHEALLY ADMINISTERED ANTIFLAMMIN-1 ON BLEOMYCININDUCED LUNG FIBROSIS Wei Liu, Jin Feng Li, Xiao Juan Shi, Chen Li, Wen Li Liu, Jian Zhong Han, Dan Dan Feng, Si Yuan Tang, Zi Qiang Luo* Department of Physiology, Xiangya School of Medicine, Central South University, Changsha 410078, P.R. China Antiflammin-1 (AF-1, MQMKKVLDS) is a nonapeptide which has powerful anti-inflammatory effect. It acts as its original full length protein-Clara Cell Secretory Protein (CCSP) and may display potently clinical application value. The previous studies indicate that bleomycin-injured mice down-regulate the expression of CCSP, and our further study has suggested Clara cell of the terminal bronchioles can significantly inhibit the collagen deposition and fibroblast proliferation in bleomycin-induced pulmonary fibrosis. In this study we investigated whether the AF-1 had a protective role against fibrosis in lung. To establish the fibrotic model, mice were subjected to intratracheal administration of bleomycin (5 mg/kg), and then received AF-1 (7.5 mg/kg) by intratracheal administration immediately. ELISA revealed that TNFa, IL-1b, TGFb levels in lung homogenates of bleomycin-injured mice with AF-1 treated decreased significantly than those of only bleomycin-administered mice at 3, 7, 14 days after bleomycin administration. The collagen deposition was quantified by Masson staining and hydroxyproline content, the fibroblast proliferation was evaluated by immunohistochemical analyses for a-Smooth muscle actin at 28 days after bleomycin administration, and these results all showed that AF-1 could reduce the degree of lung damage and fibrosis. In conclusion, Antiflammin-1 has protective effect on bleomycin-induced lung fibrosis and it might prove useful as an add-on therapy for the treatment of fibrotic disorders of the lung such as idiopathic pulmonary fibrosis, a disease that still represents a major challenge to medical treatment.
EFFECTS OF LIVER APOPTOSIS AND PROLIFERATION ON SIMULATED 4000M HILO RATS Wen Feng Liu, Shu Lin Qu, Chang Fa Tang Exercise Physiology Research Center, Hunan Normal University, ChangSha, China In this research we tried to provide research data for a medium and practice science application about hypoxia. 60 male SD rats (Hunan Agricultural University supplied, license number: XIANG scxk 2003-003) were randomly divided into six groups: control (C), 8 hours hypoxic exposure (8hHE), 12 hours hypoxic exposure (12hHE), purely training (T), 8hHilo and 12hHilo. C did not hold hypoxia and training. HE and Hilo were held in hypoxia chamber (as a altitude of 4000 m) with 8 hours and 12 hours each day. T and Hilo were trained at the speed of the 25 m/min for 1 hour each day, 5 days per week for 4 weeks.
MICRORNA-375 PROMOTES 3T3-L1 PROADIPOCYTE DIFFERENTIATION Hong Yan Ling 1,2, Xiao Yong Lei 1, Zhi Ping Gao 1, Wei Dong Yin 1, He Sheng Ou 1, Duan Fang Liao* 1 1 Division of Pharmacoproteomics, Institute of Pharmacy and Pharmacology, University of South China, Hengyang, China 2 Department of Pharmacology, School of Pharmaceutical Science, Central South University, Changsha, China MicroRNAs (miRNAs) represent a class of short (~22 nt), noncoding regulatory RNAs which are involved in multiple biological processes such as energy homeostasis, sugar and lipid metabolism, and cell differentiation by negatively regulate gene expression at the post-transcriptional level. To investigate the effect of microRNA-375 on differentiation of 3T3-L1 preadipocyte (3T3-L1 cell), 3T3-L1 cells were cultured and transfected with microRNA-375 plasmids, and were induced to differentiation using with 0.5 mmol/L 3-isobutyl1-methyxanthine, 1 mg/L insulin, and 1mmol/ L dexamethasone. Oil red O staining was performed and marker genes of preadipocyte differentiation were detected by using real-time PCR. Results showed that the microRNA375-transfected 3T3-L1 cells contained many more small lipid droplets than did non-transfected 3T3-L1 cells before induced. Following induction, differentiation in the microRNA-375-transfected 3T3-L1 cells was dramatically promoted. The expression of adipocyte differentiation marker genes such as lipoprotein lipase (LPL), adipocyte fatty acid-binding protein (aP2) and peroxisome proliferator-activated receptor gamma (PPARgamma) were upregulated in the microRNA-375-transfected cells, whereas expression of preadipocyte factor-1 (Pref-1), an inhibitor of preadipocyte differentiation, was downregulated. These results suggest that microRNA-375 could promote 3T3-L1 preadipocyte differentiation, indicating microRNA-375 may be of potential use in the treatment of obesity and obesity-induced insulin resistance.
APPLICATION OF A BACTERIAL TWO-HYBRID SYSTEM FOR THE ANALYSIS OF BRS-3 INTERACTED PROTEINS Hui Jun Liu, Yu Rong Tan, Yang Xiang, Xiao Yan Zhou, Fei Qu, Chi Liu, Xiao Ling Zhu, Xiao Qun Qin* Department of Physiology, Xiangya School of Medicine, Central South University, Changsha, 410078, China
S59
Proteineprotein interactions play an important role in virtually all cellular processes including translation and modification of proteins, living things respondent reaction, the signal transduction and a lot of metabolic process. The bacterial two-hybrid (BTH) system has become a widely used tool for determining such interactions in vivo. Since there is very little known about the biological function, we have screened the interacted proteins of BRS-3 by using BTH technology. The pBT/BRS-3 bait plasmid was constructed and verified by double digestion and sequencing. Western blot analysis confirmed that the molecular weight of recombinant fusion protein was about 70 kDa, which was unanimous with a predicted molecular weight. Since the bait protein alone cannot activate the transcription of reporter gene, it can be used for screening of fetal brain library. Interactions were determined to be positive as measured by growth on "Selective Screening Medium" and validated by growth on "Dual Selective Screening Medium". To identify the proteins encoded by the target DNAs, the nucleotide of the target DNA was sequencing and compared to nucleotide databases to identify related proteins. All together 9 kinds of proteins and 4 kinds of new genes were identified. The findings of the BTH screen were confirmed by the glutathione S-transferase (GST)-pull down assay. These results showed that BRS-3 could interact with GRP, CNTF, SERPINB5, SERPINE1, HPN, PTK6, CSNK2A1, PRKCB1 and BIRC5. These proteins have a wide range of functions including cell growth, differentiation, anti - apoptosis, cytoskeleton construction, and the tyrosine kinase activities.
PROTECTIVE EFFECT OF INTRATRACHEALLY ADMINISTERED ANTIFLAMMIN-1 ON BLEOMYCININDUCED LUNG FIBROSIS Wei Liu, Jin Feng Li, Xiao Juan Shi, Chen Li, Wen Li Liu, Jian Zhong Han, Dan Dan Feng, Si Yuan Tang, Zi Qiang Luo* Department of Physiology, Xiangya School of Medicine, Central South University, Changsha 410078, P.R. China Antiflammin-1 (AF-1, MQMKKVLDS) is a nonapeptide which has powerful anti-inflammatory effect. It acts as its original full length protein-Clara Cell Secretory Protein (CCSP) and may display potently clinical application value. The previous studies indicate that bleomycin-injured mice down-regulate the expression of CCSP, and our further study has suggested Clara cell of the terminal bronchioles can significantly inhibit the collagen deposition and fibroblast proliferation in bleomycin-induced pulmonary fibrosis. In this study we investigated whether the AF-1 had a protective role against fibrosis in lung. To establish the fibrotic model, mice were subjected to intratracheal administration of bleomycin (5 mg/kg), and then received AF-1 (7.5 mg/kg) by intratracheal administration immediately. ELISA revealed that TNFa, IL-1b, TGFb levels in lung homogenates of bleomycin-injured mice with AF-1 treated decreased significantly than those of only bleomycin-administered mice at 3, 7, 14 days after bleomycin administration. The collagen deposition was quantified by Masson staining and hydroxyproline content, the fibroblast proliferation was evaluated by immunohistochemical analyses for a-Smooth muscle actin at 28 days after bleomycin administration, and these results all showed that AF-1 could reduce the degree of lung damage and fibrosis. In conclusion, Antiflammin-1 has protective effect on bleomycin-induced lung fibrosis and it might prove useful as an add-on therapy for the treatment of fibrotic disorders of the lung such as idiopathic pulmonary fibrosis, a disease that still represents a major challenge to medical treatment.
EFFECTS OF LIVER APOPTOSIS AND PROLIFERATION ON SIMULATED 4000M HILO RATS Wen Feng Liu, Shu Lin Qu, Chang Fa Tang Exercise Physiology Research Center, Hunan Normal University, ChangSha, China In this research we tried to provide research data for a medium and practice science application about hypoxia. 60 male SD rats (Hunan Agricultural University supplied, license number: XIANG scxk 2003-003) were randomly divided into six groups: control (C), 8 hours hypoxic exposure (8hHE), 12 hours hypoxic exposure (12hHE), purely training (T), 8hHilo and 12hHilo. C did not hold hypoxia and training. HE and Hilo were held in hypoxia chamber (as a altitude of 4000 m) with 8 hours and 12 hours each day. T and Hilo were trained at the speed of the 25 m/min for 1 hour each day, 5 days per week for 4 weeks.