Role Of Endoplasmic Reticulum Stress In The Cognitive Dysfunctions Rats With Coal Worker’s Pneumoconiosis

Background and objective: Coal worker’s pneumoconiosis (CWP) is one of theimportant occupational diseases in our province in China. The serious respiratorydysfunctions caused by CWP attract increasing attention of influence of CMP on brainfunction by society. It is of great clinical value to research the reason and mechanism of theinfluence of CWP on brain function, and this is also our medical community’s obligatoryresponsibility. Studies showed that, with the promotion of CWP, the normal rate of pulmonaryfunction had sequentially reduced, following with the increasing risks of pulmonarydysfunction. As a result, it usually caused more serious pulmonary function obstacledisproportionate with damages of lung, leading to hyoxemia and hypercapnia, which causeddamages of various organs system other than respiratory system, especially an obviousinfluence on central nervous system. In the early days, it often happened that the cognitivefunction of capacities of learning, memory and calculation had changed. But the occurrencemechanism was still not entirely clear.As an important organelle to synthetize, modify, assemble and fold proteins and storeCa2+in eukaryotic cells, endoplasmic reticulum (ER) attracts more and more attention for itsrote of maintaining cell homeostasis and affecting cell survival. However, in recent years, ithas been found that the dysfunction of endoplasmic reticulum, such as lack of glucose, changeof Ca2+homeostasis, and damage of free radical, would cause proteins to fold and accumulateincorrectly in endoplasmic reticulum, and its following stress reaction of endoplasmicreticulum would lead to cell specific apoptosis. This is considered to be the third way ofapoptosis following the ways of death receptor and mitochondria. The physical and chemicalfactors inducing endoplasmic reticulum stress (ERS) include ultraviolet rays, lack of nutrientsor oxygen, oxidative stress, high homocysteine level, viruses, poisonous substances (heavymetal), etc. These factors are at work by various mechanism. On the one hand, throughvarious reaction paths endoplasmic reticulum stress could reduce the protein synthesis level,up-regulate the chaperonin expression of endoplasmic reticulum, improve the resistanceability to various stimulates and help cells to resume homeostasis as soon as possible.However, in the other hand, when the incitants persist or intensity is too large, theendoplasmic reticulum stress reaction is not enough for helping cells to resume homeostasis,so that the programmed cells apoptosis way is launched, leading to cells apoptosis.The long-term anoxic and chronic hypoxia caused by coal worker’s pneumoconiosis is important triggers to endoplasmic reticulum stress. They could activate endoplasmicreticulum stress response in nerve cells, and then lead to nerve cells apoptosis by theapoptosis way of caspase-12 as initiation factor and CHOP as informational molecule forcentral. This could cause brain dysfunction and symptoms of cognitive impairment up todementia in the early days.Our team led by Professor has done meaningful explorations in the field of coal worker’spneumoconiosis (CWP) and brain function change. We have found that higher incidence ratesof cognitive dysfunction have happened in patients with coal worker’s pneumoconiosis with aseries of animal studies. Under the guidance of endoplasmic reticulum stress theory, wesuccessfully establishing a rat model of cognitive dysfunction caused by coal worker’spneumoconiosis (CWP). This could help to discuss the pathogenesis of influence of coalworker’s pneumoconiosis (CWP) on cognitive function, providing new ideas and new strategyfor clinical prevention, diagnosis and treatment. This study is divided into two parts. The firstpart is mainly mean to establish the animal model of influence on capability of learning andmemory in rats with coal worker’s pneumoconiosis; and the second part focuses on discussingthe role of endoplasmic reticulum stress in cognitive dysfunction of rats with coal worker’spneumoconiosis.The First Part Establishment the animal model of influence on capability oflearning and memory in rats with coal worker’s pneumoconiosisMethods: 100 male Wistar rats weight 180~200g. The primary coal in Mining Bureau inJincheng City was grinded and crushed. After being filtered by 200 mesh stencils and selectedby using water, the 50 g/L of suspension was prepared using physiological saline, and thenautoclaved. Trachea cannulas were prepared by 18-size vein trocars. After disinfection on theabdomen and anesthesia by 10% hydration chlorine aldehyde (300 mg/kg.w), rats weresupinely fixed to being processed orotracheal intubation. The model group animals wereinfused into coal dust physiological saline suspension (1 ml per mouse) through theendotracheal tube, and the normal control group animals were infused into amountphysiological saline. The infusion was processed one time at the beginning. All theexperimental animals were under the same conditions of breeding and free for food. After 8weeks and 20 weeks, rats were respectively extracted from experimental group and controlgroup randomly to be determined their capabilities of learning and memory by Morris watermaze system. In sterile conditions abdominal aortic blood was extracted for blood gasanalysis, and lung and hippocampus brain tissue was gathered for morphological observation. Results:1, Results of place navigation test and spatial probe test in the Morris water maze testshowed that the capacity of learning and memory of rats in the model had significantlyimpaired, which was related to the time of dyeing dust.2, With the time of dyeing dust prolonged in the animals being perfused coal-dustthrough air passages, arterial partial pressure of oxygen (PaO2) gradually reduced whilePaCO2gradually raised, so that pulmonary function of the experimental animals weregradually reduced after dyeing dust, which was related to the time of dyeing dust.3, The results of VG dyeing showed that fibrosis indices in lung tissue presentedsignificantly higher with in the disease progression, so that dyeing dust was an importantfactor causing lung tissue fibrosis.4, Changes of pathomorphology in lung tissue: it was found under optical microscopethat there was a tremendous amount of coal dust deposition visible in lung tissue of rats dyingdust for 8 weeks. Inflammatory reaction could be found in interstitial and alveolar area, withpulmonary interstitial bleeding and evenly protein edema fluid in part of the alveolar space.There were damages of lung tissue, interstitial fibre hyperplasia, and decompensatedemphysema visible in part of this area.There still was much coal dust deposition in lung tissue of rats dying dust for 20 weeks.Huge macrophages had phagocytized the coal dust were visible, as well as edema, thickeningsof alveolar walls in lesions area and obvious interstitial fibre hyperplasia. Alveolar walls weredamaged or significantly squeezed in decompensated emphysema area, with multiplecoal-dust nodules visible.5, Changes of pathomorphology in brain tissue: it was found under optical microscopethat there was obvious inflammatory reaction in rats dying dust for 8 weeks. It showedangiectasis, congestion, gliocyte increasing, interstitial loose, nerve cells neurite and nucleolidisappearance, indicating that there were edema and nerve cells denaturation in the braintissue. Part of the neuron chromatin was concentrated and a few apoptotic body was visible.Denaturation of nerve cells thickening of nuclear membrane, chromatins condensed intoblocks and more evident karyopyknosis were accentated in rats dying dust for 20 weeks. Andthe apoptotic bodies increased compared with that in rats dying dust for 8 weeks. Themesenchyme had a structure of screen, indicating that there were focal liquefactive necrosisand karyorrhexis and necrotic cells were visible.6, Neuron specific enolase (NSE) dyeing count showed that NSE highly specificallylocates in neurons, the number of nerve cells of coal worker’s pneumoconiosis group is obviously less than the control group, indicating the important mechanism leading tocognitive dysfunction.The Second Part Role of endoplasmic reticulum stress in the cognitivedysfunctions rats with coal worker’s pneumoconiosisMethods: Male Wister rats weighing 180~200g were provided by Experimental AnimalCenter of Shanxi Medical University. Had been adaptability fed for one week, The modelgroup animals were infused into 1 ml of coal dust physiological saline suspension (50g/L) permouse through the endotracheal tube, and the normal control group animals were infused into1 ml of physiological saline per mouse. The infusion was processed one time at the beginning.All the experimental animals were under the same conditions of breeding and free for food.After dyeing dust for 8 weeks, animals were narcotized by 10% hydration chlorine aldehyde(300 mg/kg.w) through intraperitoneal injection. The brain tissue was removed and part of thetissue was stored in liquid nitrogen. The content of Malondialdehyde (MDA) of preparedhomogenate was tested by biochemical process. Part of the tissue was fixed in 10% of theneutral formaldehyde for histologic study. The protein expression of HIF-1α, GRP78, CHOP,caspase-12 and Bcl-2 was observed by immunohistochemical staining method.Semi-quantitative analysis was processed to positive staining area by BI2000 microscopicimage analysis system under optical microscope for positive staining area for half quantitativeanalysis. The apoptotic cells were stained by TUNEL method, and the positive staining cellswere counted by BI2000 microscopic image analysis system.Results: Compared with normal control group, the protein expression of HIF-1α, GRP78,CHOP, caspase-12, Bcl-2 significantly increased in model rats, and so did the positive rates ofapoptotic cells. The obviously increased apoptosis of signs were observed by morphology,fully explaining that hyoxemia in rats with coal worker’s pneumoconiosis for 8 weeks hadcaused endoplasmic reticulum stress response in the brain and neural cells apoptosis.As above studies results shown, the significance of this study is as follows: (1) A ratmodel on cognitive dysfunction caused by coal worker’s pneumoconiosis was successfullyestablished, providing an important method and means for related research. (2) The importantsignificance of endoplasmic reticulum stress mechanism on the cognitive dysfunction wasfound, deepening the research mentality, broadening the research filed, and bringing forth newideas on the theory of pathogenesis.