| Nerve block is an anesthesia method which can analgesic complete, easy to operate and has small perturbation in the vital signs of patients. Furthermore its side effects and complications are much lower than general anesthesia, so it is widely used in clinical anesthesia and postoperative pain therapy and so on.The most commonly used drug of nerve block anesthesia is a local anesthetic, which works by blocking nerve impulses to achieve the effect block.However, local anesthetic has neurotoxicity. Especially when high concentrations or prolonged use of local anesthetics, it may cause neurotoxicity damage resulting neurological dysfunction. This caused clinical workers and researchers pay close attention to it. There is an epidemiological data show that: 30% of patients after spinal anesthesia may occur transient neurological syndrome; and serious nerve damage complications such as cauda equina syndrome whose incidence of cauda equina syndrome up to 1/8238-1/2834.Although a large number of domestic and international scholars and research reports:local anesthetic will be neurotoxicity, but the exact mechanism has not yet been clarified.Our preliminary findings indicate that:Local anesthetics can reduce the SH-SY5Y cells ATP Generation, activate MAPK signaling pathway,and cause an increase in intracellular ROS, eventually leading to nerve cell apoptosis. Local anesthetics can also activate the cell membrane T-type calcium ion channels, which triggers intracellular calcium overload and promote apoptosis, ultimately causing neurotoxicity damage.In addition, the study we found that local anesthetics can lead to SH-SY5Y cell DNA damage,which will lead to cell apoptosis and necrosis.DNA is life’s most important genetic material, DNA stability affect the survival and development of the organism.But the integrity of the organism’s genes susceptible to external environmental factors,radiation and chemicals can damage the structure of the DNA double.Evidence suggests that oxidative stress/ROS is an important factor which can lead to cell DNA damage. Our previous studies have confirmed that the outbreak of intracellular ROS is an important mechanism for the local anesthetic to cause neurotoxicity. Therefore, local anesthetics may also by stimulating ROS burst causing DNA damage in nerve cells, eventually causing neurological complications.The type of DNA damage are many and varied, while DNA damage will activate related DNA repair signal pathways.The type of injury oxidative stress/ ROS-induced cell DNA is mainly oxidized base damage.Such oxidative damage mainly be repaired by excision repair mechanism, such as:DNA base excision repair (BER) and nucleotide excision repair (NER).Our previous studies show:local anesthetics can cause SH-SY5Y cells DNA damage and in SH-SY5Y cells after injury the critical repair enzyme of nucleotide excision repair pathway, the XPD (xeroderma pigmentosum complementation group D) expression levels increased.Local anesthetics may cause many types of DNA damage in nerve cells, and these different types of DNA damage will be repaired at the same time repair by a variety of mechanisms.Currently repair pathways can be divided into the following categories:base excision repair (BER), nucleotide excision repair (NER), mismatch repair (mismatch), recombination repair (recombination).What is the relationship between nerve cell DNA damage caused by local anesthetics with the above-described DNA repair pathways with?There have not been reported in domestic and international.Results of Our cDNA microplate genetic screening and iTRAQ proteomic screening showed:After bupivacaine treated nerve cells, nucleotide excision repair enzyme XPD, base excision repair enzyme PARP1 expression was significantly increased. It is reasoned that the two pathways may be an important way to repair local anesthetic nerve cells induced DNA damage.To demonstrate this hypothesis, this study intends to use in vitro cell experiments and animal studies in two parts, uses a variety of molecular biology techniques and discusses from the following four chapters:Chapter I Nerve cell toxicity and DNA damage caused by bupivacaineObjective established the cellular and a rat model about bupivacaine cause neurotoxicity damage and DNA damage. Discussion bupivacaine induced nerve cell toxicity and DNA damage.Methods 1.Cell ModelFirst treated SH-SY5Y cells with different concentrations of bupivacaine,CCK-8 assay cell viability and calculate the IC50 value of bupivacaine, LDH detect cytotoxicity of each group,SH-SY5Y cell were treated with IC50 values of bupivacaine for 3 hours,than change the nomal medium and incubated for 24 hours sa the cell model group. Hoechst-33258, TUNEL method detects cell apoptosis,JC-l detects mitochondrial membrane potential,DHE detects intracellular oxidative stress, Comet assay detects cell DNA damage,Western blot assay of apoptosis-related proteins Bax/Bcl-2 ratio, cleave-caspase3 expression, protein expression of p-γH2ax (DNA damage markers).2.Animal modelWe choose the SD rats after intrathecal catheter, injection of 3% bupivacaine as an animal model of injury.Spinal cord tissue HE staining to observe pathological changes of neurons,ELISA detection of oxidative damage malondialdehyde (MDA) and oxidative DNA damage markers 8-hydroxy-deoxyguanosine (8-oxodG),TUNEL assay and apoptosis related protein Bcl-1/Bax ratio detect neuronal apoptosis in spinal cord tissue,Western blot detected of phosphorylated levels of protein -γH2ax indicated DNA damage of rat spinal cord tissue,Stimulate latency (PWTL) by foot photothermal detection rats, mechanical pain paw withdrawal threshold (PWMT) mean the animal sensitivity to heat pain, mechanical pain stimulus;Used the behavioral Indicators analyze rat spinal cord function by the damage.Statistical Method Results are expressed as the mean ± standard deviation (SD). Data analysed by SPSS 13.0 software for statistical treatment. The Student’s t test or one way ANOVA with repeated measure was used for statistical analyses of data where applicable. Multiple comparisons tests were performed by LSD (homogeneous variance) or Dunnett’S T3(heterogeneity of variance) Statistical significance was set at P value less than 0.05.Results CCK-8 assay cell viability results show:After the cells were treated with different concentrations(0、0.5、1.0、1.5、2.0、2.5 mM) of bupivacaine, bupivacaine IC50 value (half inhibitory concentration)= 1.5mM, LDH assay showed:Compared with the control group, the group started from Bup 1.5mM cytotoxic concentration was significantly higher (* p<0.05), bupivacaine cytotoxicity in a dose-dependent manner;Hoechst-33258, TUNEL detect apoptosis of each group, the results showed: 1.5mM bupivacaine treated cells may induce apoptosis SH-SY5Y ratio increase (* p <0.05);Apoptosis related protein Bcl-2/Bax ratio decreased and apoptosis proteins cleave-caspase3 increased expression (* p<0.05);JC-1 detected the mitochondrial membrane potential, the results show:bupivacaine treated SH-SY5Y cells significantly decreased mitochondrial membrane potential (* p<0.05), leading to mitochondrial damage;after SH-SY5Y treated by bupivacaine significantly increased positive DHE (* p<0.05), showing an increase of intracellular ROS;Comet Assay relevant indicators (comet tail moment) increased (* p<0.05), protein phosphorylation level yH2ax increased (* p<0.05) indicate that after treated by bupivacaine SH-SY5Y cells DNA damage was significantly increased.HE spinal cord tissue showed that the control group of rat spinal cord neurons morphology, structure normal.6 h group,12 h group and 24 h group showed nuclear condensation, fragmentation, dissolution, degeneration and necrosis or disappear;the nerve cell degeneration, necrosis of the number of 24 h group were significantly more than the other groups;MDA test results showed that:compared with the control group, MDA content in model group was significantly increased, the difference was statistically significant (P<0.05);TUNEL assay showed:Compared with the control group, the model group rat spinal cord neuronal apoptosis was significantly increased, the difference was statistically significant (P<0.05);Spinal cord tissue apoptosis related protein Bcl-2/bax expression ratio values were significantly lower, the difference was statistically significant (P<0.05);ELISA detected 8-oxo-dG content, results showed that:compared with the control group, model group, 8-oxo-dG were significantly increased, the difference was statistically significant (P <0.05);Compared with the control group, model group spinal cord yH2ax protein phosphorylation levels were significantly increased, the difference was statistically significant (P<0.05);Behavioral Results:Compared with the control group, model group mechanical pain paw withdrawal threshold (PWMT) and reflexology photothermal stimulation of rat latency (PWTL) values were increased, the difference was statistically significant (P<0.05).Conclusion This chapter experiments confirmed:bupivacaine can cause neurotoxicity damage, as follows:the proportion of apoptosis increased apoptosis-related protein expression increased, intracellular levels of oxidative stress increased, mitochondrial membrane potential, DNA damage increased.Chapter II Gene and proteomic screening:investigate the key enzymes expression of associated DNA damage repair pathways after neurons DNA damage induced by local anestheticsObjective To investigate the key enzymes expression of associated DNA damage repair pathways after neurons DNA damage induced by local anesthetics.Methods First, we established the SH-SY5Y cell damage model,according to the chapter I. cDNA screening microplate array technology was used to detected a number of important regulatory factor and repair enzymes of the DNA damage repair pathways,iTRAQ proteomic screening technology was used to detected differentially expressed proteins between local anesthetic nerve injury model group (group B) and control group (group C), especially in the differentially expressed proteins of the DNA repair associated pathways.Statistical Method Results are expressed as the mean ± standard deviation (SD). Data analysed by SPSS 13.0 software for statistical treatment. The Student’s t test or one way ANOVA with repeated measure was used for statistical analyses of data where applicable. Statistical significance was set at P value less than 0.05.Results cDNA microplate results showed that:Compared with control cells, after being treated by bupivacaine the differentially expressed genes are as follows: DNA-PKcs, PTEN, NTH1, RAD9, CSB, GADD45, XPD, XPC-HR23B, P53; Furthermore, these repair genes are mainly concentrated in the following three repair pathways:1. base excision repair (BER); 2. nucleotide excision repair (NER); 3. non-homologous recombination repair (NHEJ).iTRAQ proteomic screening results showed that:compared with the control group, nerve cells after DNA damage caused by bupivacaine activated repair enzymes are mostly concentrated in the base excision repair pathway (pol8, plo(3, PARP) and nucleotide excision repair pathways (HR23B, RFC, po1δ, XPD).Conclusion Through the cDNA microplates and iTRAQ proteomic screening, we found that after the local anesthetic nerve cells caused DNA damage repair differentially expressed proteins mainly concentrated in the following two repair mechanisms:1.base excision repair (BER); 2. nucleotide excision repair (NER).Chapter ID Verify the possible major repair pathways and differential expression of repair enzymesObjective Based on the results of the previous chapter of the gene and proteomic screening,to verify the possible major repair pathways (BER, NER) and differential expression of repair enzymes:XPD> PARP-1Methods 1. Cell ModelAfter 1.5mM bupivacaine treated SH-SY5Y cells, crucial DNA repair gene (XPD, PARP-1) mRNA and protein expression were observed on 3h,6h,12h,24h at different time points.Q-PCR verified the mRNA expression of repair gene XPD、 PARP-1 in DNA damage cell model caused by bupivacaine.Western blotting detected the key repair enzyme XPD and PARP-1 expression in DNA damage cell model caused by bupivacaine.2. Animal ModelThe animal nerve injury model were established firstly(according to the first chapter).Western blotting and immunohistochemistry (IHC) detected "critical repair enzyme" XPD and PARP-1 expression in the rat spinal nerve DNA damage model.Statistical Method Results are expressed as the mean ± standard deviation (SD). Data analysed by SPSS 13.0 software for statistical treatment. The Student’s t test or one way ANOVA with repeated measure was used for statistical analyses of data where applicable. Statistical significance was set at P value less than 0.05.Results 1. Cell Modelq-PCR detect the expression of mRNA of XPD and PARP-1 results showed: Compared with control cells, after treated by bupivacaine the expression of XPD and PARP-1 mRNA were increased at different time, the difference was statistically significant (P<0.05);Western blotting detected the protein expression of XPD, results showed:Compared with control cells, the protein expression of XPD enzyme are increase at different time points after treatment bupivacaine;But there is no significantly increassion at 3h group(P> 0.05), the other time points XPD protein expression were statistically significant increase (* P<0.05);there is no significantly increase at 3h group(P> 0.05), the other time points PARP-1 protein expression were statistically significant increase (* P<0.05);2. Animal ModelWestern blotting detected the protein expression of XPD and PARP-1, results showed:compared with the control group, XPD and PARP-1 protein expression were significantly increased, the difference were statistically significant (* P<0.05) in model group rat spinal cord tissue; IHC detected the protein expression of XPD and PARP-1, results showed:compared with the control group, XPD and PARP-1 protein expression were significantly increased, the difference were statistically significant (* P<0.05) in model group rat spinal cord tissue;Conclusion Nucleotide excision repair pathways (key enzyme XPD) and base excision repair pathways (key enzyme PARP-1) are activated, after bupivacaine leading to neuronal DNA damage, and this result consistent with cDNA microplates and iTRAQ proteomic screening results.Chapter IV Verify the functionality of the possible major repair pathways and differential expression of repair enzymesObjective Based on the results of the previous chapter of the gene and proteomic screening,to verify the functionality of the possible major repair pathways (BER, NER) and their differential expression of repair enzymes:XPD、PARP-1Methods Through the XPD low expression lentivirus infection SH-SY5Y cells to established low XPD expression of SH-SY5Y cell line;Using comet assay and Western blotting of DNA damage markers protein phosphorylation levels of y-H2ax to evaluate the repair of DNA damage of this model;LDH detected cytotoxicity, Annexin V-FITC/PI flow cytometry detected the percentage of apoptotic cells in each group, Western blotting detected the exppression of apoptosis-related proteins;Before established bupivacaine cause nerve cell toxicity model, we used PARP-1 inhibitors 20nM-PJ34 pretreatment SH-SY5Y cells,using comet assay and Western blotting of DNA damage markers protein phosphorylation levels of y-H2ax to evaluate the repair of DNA damage of this model;LDH detected cytotoxicity, Annexin V-FITC/PI flow cytometry detected the percentage of apoptotic cells in each group, Western blotting detected the exppression of apoptosis-related proteins.Statistical Method Results are expressed as the mean ± standard deviation (SD). Data analysed by SPSS 13.0 software for statistical treatment. The Student’s t test or one way ANOVA with repeated measure was used for statistical analyses of data where applicable. Statistical significance was set at P value less than 0.05.Results The mRNA and protein expression levels of XPD were significantly lower (* p<0.05) after XPD-RNAi (31535) lentivirus group infected the SH-SY5Y cells;Compared with the model group, low expression of XPD-SH SY5Y cells group after treated by bupivacaine, DNA damage was aggravated, comet assay-related injury indicators tail moment (Olive tail moment) was significantly higher (* p <0.05);DNA damage markers protein y-H2ax phosphorylation levels were significantly increased (* p<0.05); flow cytometry apoptosis was significantly increased (* p<0.05); LDH Cytotoxicity detected injury was aggravated (* p<0.05); the expression of apoptosis-related protein Bax increased (* p<0.05);With the model group (Bup group) comparison, PJ34+Bup group significantly increased DNA damage, comet assay-related injury indicators tail moment (Olive tail moment) was significantly higher (* p<0.05);DNA damage markers protein y-H2ax phosphorylation levels were significantly increased (* p<0.05); the proportion of apoptosis of the PJ34+Bup group was detected by flow increased significantly (* p <0.05); LDH Cytotoxicity detected injury was aggravated (* p<0.05); the protein expression of Bax increased (* p<0.05).Conclusion After bupivacaine-induced cell DNA damage, the key repair enzyme XPD of nucleotide excision repair pathway and the key repair enzyme PARP-1 of base excision repair pathway were involved in repairing the damage. |
PDF Full Text Request