| Background and objectiveLocal anesthetics neurotoxicity and position causing obstetric neurological complications has been caused concern, especially gestational diabetes maternal have the susceptibility to anesthetic nerve toxicity. The major manifestation is enhanced susceptibility in gestational diabetes mellitus to neurotoxicity of local anesthetic agents. It has been demonstrated that after intraspinal anesthesia or analgesic therapy in diabetic patients, the risk of neural damage is significantly higher. Or it may cause the existing polyneuropathy to deteriorate.Bupivacaine is one of the common local anesthetic, and the reports about the toxicity reaction of nerves and cardiovascular system induced by bupivacaine increased gradually,which attract people’s utmost attention.but the mechanism is not very clear, especially the neurotoxic effects.Another research find that Bupivacaine may induce Schwann cells generate a quantity of reactive oxygen species and activate the enzyme of Caspase-3to trigger the apoptosis of neurons.It is not clear whether the mechanism in pregnant women with GDM is consistent with normal pregnant women. Endoplasmic reticulum stress plays an important pathophysiological role to the susceptibility of local anesthetical neurotoxicity in pregnant women with GDM. ER stress is one of the main factors that pregnant women with GDM are susceptibile to local anesthetical neurotoxicity. The researchs of diabetic rats induced by STZ (streptozotocin) have directedly confirmed that a higher level of ROS in neurons may occur in diabetes mellitus or high sugar enviorment.Local anesthetics disturb the function of endoplasmic reticulum, and eventually lead to stress on organelle and mitochondrial dysfunction. Then reactive oxygen species is produced. A higher level of ROS in cells is a positive feedback to ER stress, enhancing ER stress. Hence chronic ER stress will facilitate the toxic accumulation of ROS in cells, leading to apoptosis. By this,we can speculate that patients withdiabetes or gestational diabetes in high sugar environment together with local anaesthetic effect to trigger ROS.Endoplasmic reticulum sngnals on multiple links and formate function coupling with other organelles. Mitochondria is one of the most important organs inducing apoptosis in ER stress. Due to the omentum of endoplasmic reticulum and the membrane of mitochondria contact closely, any small disturbance in endoplasmic reticulum can affect the function of mitochondria. Under the condition of overnutrition or metabolic regulation signal obstructed, as the site of synthesis and processing about protein and lipid, endoplasmic reticulum overload and lead to ER stress. Through various channel to damage mitochondrial DNA and reduce the number of mitochondria,the function of mitochondrial is impaired. So it lead to apoptosis. Triggering neural cell apoptosis may be implemented by way of multiple loci and the role.Our team’s preliminary experiments found that local anesthetics act on SH-SY5Y cells injury model and in high sugar training set of local anesthetics intracellular ROS content and cell apoptosis rate increased significantly.High sugar environment may make neuron intracellular and extracellular environment abnormal. It is adversely to neuron physiological activity, especially to membrane system that are suspectible to the abnormal. Osmotic pressure of high sugar enviroment often unusually fluctuating in the abnormal physiological conditions,it may impaire function of cell membranes and organelles,then the impaired cell membranes and organelles are vulnerable to ROS. Chronic high sugar cause mitochodria to produce a quantity of ROS, then make organelles and membrane and nucleus progressively impaired, at last the neurons have been damaged.Glucose regulatory proteins (Grp) can respond in a timely manner to changes of intracellular and extracellular environment. Grp is a kind of endoplasmic reticulum stress proteins in cells, belonging to the family of SHP90, which reside in the endoplasmic reticulum lumen,called endoplasmic reticulum molecular chaperone, with many important biological functions. At the early stage of ER stress,Grp78is one of important member to resist the stress,increasing cell viability in the adversity,mainting endoplasmic reticulum steady state and preventing apoptosis.This study bases on the changes of endoplasmic reticulum of neuron cells in high sugar environment and the apoptosis of neuron cells to prove the acculumation of ROS in ER stress. Once the use of local anesthetic, a higher level of ROS in cells is a positive feedback to ER stress, enhancing ER stress and increasing apoptosis. The study not only put forward a new theory of molecular mechanism about neurological complications occurring in some parturient women with GDM or diabetic patients after intraspinal anesthesia having increasing incidence,but also supply with a new clue of pathogenesis about neurological complications occurring in some parturient women with GDM having increasing trend. At the same time, the study propose new target to cure neurological complications.The study construct lentiviral vector recombinant plasmid with over expression gene Grp78, on the other hand, the study construct lentiviral vector recombinant plasmid with over expression gene Grp78knocked-down. then respectively infect SH-SY5Y cells. We regulate the expression of gene Grp78to research whether local anesthetic Bupivacaine cause neuron cells to accumulate a large quantity of ROS and to apoptosis.and we further confirm that by regulating over expressing gene Grp78can lessen ER stress to reduce the accumulation of ROS and apoptosis.Chapter1High glucose culture SH-SY5Y cells increasing ROS and apoptosisobjective To discuss SH-SY5Y cells in high sugar environment increase intracellular ROS and apoptosis.Methods we adopt different concentrations of glucose medium to culture SH-SY5Y cells,and employ flow cytometry to detect the quantity of ROS in the cells and the situation of apoptosis.we use MTT method to detect cell survival rate.At last we use Western blotting technique to detect the change of the related protein about GRP78.Measurement data were presented as mean±standard deviation. SPSS13.0software was used for statistical treatment. The quantity of ROS and the situation of apotosis and cell survival rate and the quantity of GRP78were analyzed by one-way ANOVA and comparisons among groups were made by LSD (homogeneous variance) or Dunnett’s T3(heterogeneity of variance). P<0.05was considered statistically significant.Results The quantity of ROS in the cells cultured by glucose medium increasing gradually and the apoptosis and cell survival rate have statistical differences (P<0.01).Western blot results show that GRP78express enhance in endoplasmic reticulum stress.Conclusion Intracellular ROS increasing and apoptosis are associated with endoplasmic reticulum stress caused by high sugar environment. Chapter2Bupivicaine bring about SH-SY5Y cell cultured by high-glucose increasing ROS and apoptosisobjective To discuss Bupivacaine act on SH-SY5Y cells in high sugar environment increasing ROS and apoptosis.Methods we adopt1mmol/L bupivicaine to SH-SY5Y cells cultured by different concentrations of glucose medium,and employ flow cytometry to detect the quantity of ROS in the cells and the situation of apoptosis.we use MTT method to detect cell survival rate.At last we use Western blotting technique to detect the change of the related protein about GRP78.Measurement data were presented as mean±standard deviation. SPSS13.0software was used for statistical treatment. The quantity of ROS and the situation of apotosis and cell survival rate and the quantity of GRP78were analyzed by one-way ANOVA and comparisons among groups were made by LSD (homogeneous variance) or Dunnett’s T3(heterogeneity of variance). P<0.05was considered statistically significant.Results The quantity of ROS in the cells cultured by glucose medium increasing gradually and the apoptosis and cell survival rate have statistical differences (P<0.01).Western blot results show that GRP78express enhance in endoplasmic reticulum stress.Conclusion Intracellular ROS increasing and apoptosis are associated with Bupivacaine further strengthening endoplasmic reticulum stress. Endoplasmic reticulum stress cause ROS outbreak in cells, leading to cell damage and apoptosis. With the function of GRP78degrading, endoplasmic reticulum stress further strengthen. Chapter3Cytotoxicity study of SH-SY5Y cells cultured in high glucose and treated by bupivacaineObjective To discuss the cytotoxicity of SH-SY5Y cells cultured in high sugar environment and treated by Bupivacaine.Methods SH-SY5Y cells were cultured in different concentrations of glucose and then treated by lmmol/L bupivacaine for6hours. Flow cytometry detection was performed of ROS production and apoptosis in each group of cells. MTT method was combined to detect the cell survival. Finally, Western blotting technique was used to detect the changes of GRP78in neutron cells.Measurement data were presented as mean±Standard deviation. SPSS13.0software was used for statistical treatment. The quantity of ROS and the situation of apotosis and cell survival rate and the quantity of GRP78were analyzed by two-way ANOVA and comparisons among groups were made by LSD (homogeneous variance) or Dunnett’s T3(heterogeneity of variance). P<0.05was considered statistically significant.Results ROS production detection, cell survival detection and Western blotting detection of GRP78expressions in all groups showed that P<0.01for effect of concentrations, effect of anesthetic agents as well as for the interaction between concentrations and anesthetic agents in statistical significance.Conclusion High sugar environment bring about endoplasmic reticulum stress, with ROS increasing and apoptosis adding.The usage of Bupivacaine further strengthen endoplasmic reticulum stress, causing ROS outbreak. So cells damage and apoptosis. Chapter4Construction, packaging and identification of lentiviral vector with over expression gene Grp78Objective To discuss the construction,packaging and identification of lentiviral vector with overexpression gene Grp78.Methods Using lentiviral vector GV261and genetic engineering technology to obtain the aim gene fragment, construct recombinant plasmid,prepare competent cell, transform.Through positive clone sequencing, lentivirus is packaged and virus titer is tested.Results Positive cloning sequencing comparison results show that the test pass. Melt curve did not appear mixed peak,also does not appear abnormal peak broadening. That does not appear polluted and does not appear primer dimer,also does not emerge nonspecific amplification..Conclusion Endoplasmic reticulum stress is closely related to the functional impairment of GRP78. The construction and packaging of lentiviral vector with overexpression gene Grp78give a good research tool to study the function of Grp78in endoplasmic reticulum stress. So we can further argue the function of Grp78to endoplasmic reticulum stress.Chapter5exogenous screening targets and endogenous confirmation of RNAi lentivirusObjective To discuss the construction of lentiviral vector recombinant plasmid with overexpression gene Grp78knocked-down.Methods Using RNAi lentiviral vector and over expression aim gene vector to obtain effective vector with exogenous screening targets.Through Real time PCR to confirm the endogenous aim gene knocked-down.Result The target of HSPA5-RNAi(8747-2) has the effect to knock-down the expression of the aim gene. Conclusion Endoplasmic reticulum stress is closely related to the functional impairment of GRP78. The construction and packaging of lentiviral vector recombinant plasmid with overexpression gene Grp78knocked-down give a good research tool to study the function of Grp78in endoplasmic reticulum stress. So we can further argue the function of Grp78to endoplasmic reticulum stress.Chapter6Cytotoxicity study of SH-SY5Y cells with Grp78gene over expression/knocked-down cultured in high glucose and treated by bupivacaineObjective To discuss the role of Grp78in endoplasmic reticulum stress.Methods SH-SY5Y cells with Grp78gene overexpression/knocked-down were cultured in different concentrations of glucose and then treated by lmmol/L bupivacaine for6hours. Flow cytometry detection was performed of ROS production and apoptosis in each group of cells.Measurement data were presented as mean±standard deviation. SPSS13.0software was used for statistical treatment. The quantity of ROS and the situation of apotosis were analyzed by multi-way ANOVA and comparisons among groups were made by LSD (homogeneous variance) or Dunnett’s T3(heterogeneity of variance). P <0.05was considered statistically significant.Results ROS production detection and cell survival detection, over expressing Grp78gene SH-SY5Y cells less than normal SH-SY5Y cells (P<0.01). In constract,Grp78gene knocked-down SH-SY5Y cells more than normal SH-SY5Y cells.Conclusion Enhancing SH-SY5Y cells expression of Grp78gene approach is to control high sugar disease effective method for susceptibility to neurotoxicity of local anesthetic. Overexpressing Grp78gene SH-SY5Y cells have better protective effect in endoplasmic reticulum stress,and Grp78gene knocked-down SH-SY5Y cells lack of protection in endoplasmic reticulum stress. |
