| Acute stress response is a stress disorder caused by severe mental stimulation or persistent distress,which often causes psychological and physiological problems in patients.At present,therapists mainly use psychological interventions to treat such diseases,but this treatment does not solve the physical disease problems caused by stress,and this method cannot prevent and protect stress.In view of the mental illness of adolescents with abrupt changes in adolescence and the middle-aged and elderly population under stress,sub-health problems and diseases such as insomnia,varying degrees of depression,affective disorder and schizophrenia due to acute stress have gradually increased.It has become a major problem that can not be ignored and endangers people's health.Therefore,it is extremely important to find the cause of stress disorder and to intervene and treat it as soon as possible to find a new drug with high efficiency and low toxicity.Cannabinol is extracted from plant cannabis,which has certain anti-anxiety and anti-stress effects but no toxic side effects of cannabis-like addiction.Therefore,cannabidiol has important development potential in the development of anti-stress products.In the previous study,the research team used the water-immersion restraint rat stress model in low temperature environment,and found that the acute stress rats in the acute stress rats showed obvious edema inflammation,degeneration and necrosis.To study the pharmacological effects of cannabidiol on stress-induced lung injury and to elucidate its mechanism of action,it can lay a theoretical foundation for the development of new anti-stress lung injury new drugs and functional foods.In this paper,the anti-stress effect and mechanism of cannabidiol are studied in the following aspects:Part ?:The model of lung injury in rats with acute stress was established by low temperature(20?)water immersion restraint,and the successfully modeled acute stress rats were randomly divided into the following groups:blank control group,model group,CBD experimental-L,CBD experimental-M and CBD experimental-H group(12,36,108 mg/kg,ig),and to explore the pharmacological effects of cannabidiol against acute stress-induced lung injury,it was found that:(1)The organ coefficient and wet weight/dry weight ratio(W/D)of the animal tissues in the model group were higher than those in the normal group.Compared with the model group,the lung organ coefficient and W/D value of the cannabisdiol medium and high dose groups were significantly decreased in a dose-dependent manner.This indicates that a moderate dose(36 mg/kg)of cannabinol can show a certain degree of acute damage against pulmonary edema.(2)Our group used ELISA kit to detect the levels of cytokines such as corticosterone(CORT),TNF-a and IL-1? in rat serum.The results showed that the levels of CORT,TNF-a and IL-1? in the serum of the model group were significantly higher than those in the blank group(p<0.01).However,the levels of CORT,TNF-? and IL-1? in the serum of the medium and high doses of cannabidiol were significantly lower than those in the serum of the model group(P<0.05).This indicates that acute stress can promote the production and release of a large number of inflammatory cytokines in the body,while cannabidiol produces an anti-stress effect by inhibiting the release of inflammatory cytokines in the body.(3)We performed histopathological examination.The results of HE staining showed that the lung tissue edema and alveolar wall of the model group were significantly thickened,and the alveolar structure was deformed and collapsed with a large amount of inflammatory cell infiltration.However,the cannabisdiol dose group inhibited cell infiltration and alveolar structure changes in a dose-dependent manner,and inhibited the pathological features of remodeling such as fibrosis.We successfully established an animal model of lung injury in acute stress rats by histological HE staining and cytokine detection.It also suggests that medium and high doses of cannabidiol can inhibit the production of inflammatory cytokines and pathological changes such as acute pulmonary edema,inflammatory changes and structural damage,which provides a scientific basis for the prevention and treatment of chronic obstructive pulmonary disease(COPD)Part ?:Our group found that cannabidiol has significant pharmacological effects against lung injury in rats with acute stress.In this part,our group systematically studied the pharmacological mechanism of lung injury in rats with acute stress by in vitro experiments:we first established TBHP(t-butyl hydroperoxide)induced oxidative stress in alveolar type II epithelial cells A549 Damage model.The blank group,the model group,and the cannabidiol administration group(1.25,2.5,5 ?mol/L)were set up for the following in vitro cell experiments:(1)First,we determined the cell viability by MTT colorimetry,determined the concentration of TBHP(200 ?mol)used for cell modeling,and the non-toxic dose of cannabidiol.According to the experiment,the cell survival rate of the model group was 55.3%.In contrast,cannabidiol could interfere with the cell damage induced by TBHP in a dose-dependent manner.The cell survival rates were as follows:60.5%(1.25?mol/L),73.4%(2.5?mol/L)and 82.2%(5 ?mol/L).(2)After confirming the anti-oxidative stress of cannabinol on TBHP-induced cell injury,the anti-apoptotic mechanism was systematically studied.We used Hoechst 33258 staining for qualitative observations to find that the drug exerts anti-apoptotic effects in a concentration-dependent manner.The results of nuclear staining showed that 5 ?mol/L of cannabidiol could play a significant protective role.(3)We used flow cytometry for quantitative analysis,and detected apoptosis and ROS production rate in each group by Annexin V/PI double staining and DCFH-DA staining.The results showed that 5?mol/L cannabidiol significantly reduced the intracellular reactive oxygen species induced by TBHP,and its apoptotic rate of 12.88%was significantly lower than that of the model group of 34.5%.This suggests that cannabidiol may play an anti-apoptotic role by antagonizing the high levels of reactive oxygen species caused by TBHP.Part ?:In order to explore the molecular pharmacological mechanism of cannabinol in the lung injury of rats with acute stress,our group first selected the stress rats by transcriptome sequencing technology combined with gene difference analysis and KEGG functional enrichment analysis.The expression of p38MAPK and its downstream related proteins was detected by Western Blot:(1)Molecular mechanism study in vitro:We used Western blotting to detect the expression of p38MAPK signaling pathway.It was found that cannabidiol can produce anti-apoptotic effects by inhibiting the expression levels of p-p38MAPK,Bax and Cleaved-Caspase3,and simultaneously up-regulating the expression of Bcl-2 protein.(2)Study on the molecular mechanism of animal in vivo:We further used Western blotting to detect key proteins in lung tissue.It was found that 108 mg/kg of cannabidiol can down-regulate p-p38MAPK,Bax and Cleaved-Caspase3 proteins,and up-regulate the expression level of Bcl-2 protein to counteract apoptosis induced by acute stressIn summary,cannabidiol has protective effects on lung injury induced by acute stress in rats.Its protective mechanism involves inhibiting the activation of the p38MAPK stress pathway,down-regulating the expression of Bax and Cleaved-Caspase3 proteins,thereby reducing oxidative stress damage and reducing the release of inflammatory substances.This suggests that cannabidiol has a better protective effect against stress-related diseases and provides a theoretical basis for the development of new anti-acute pulmonary stress injury drugs and functional foods. |
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