| BackgroundAcute lung injury (ALI), the causative factor of acute respiratory distress syndrome, is often characterized by numerous events, such as severe hypoxemia, alveolar-capillary barrier damage, pulmonary inflammation, non-cardiogenic lung edema and multiple organ failure. Direct and indirect injuries of the lungs are considered as the main etiological factors in the onset of this disease. Pneumococcal infection, release of gastric substances, sepsis, major trauma and acute pancreatitis are inducing factors for development of ALI. Studies have indicated that around 190,000 cases of ALI were reported in the USA, and it results in the death of 74,500 patients each year. The nuclear factor-κB (NF-κB) pathway plays a pivotal role in the pathogenesis of ALI. NF-κB is found in the cytoplasm of unstimulated cells in a dormant form bound to its inhibitor, IκBα The activation of NF-κB and its translocation from the nucleus, for binding to its associated DNA attachment site from its bound form, is triggered by external stimuli. Thus, after its release in free form, NF-κB regulates the expression of numerous genes, and results in inflammation in ALI. Despite the high morbidity and mortality associated with ALI, there are no effective therapeutic options for its treatment. Therefore, the development of novel therapies, particularly those that target NF-κB, is urgently needed. Plant or plant-based products have received considerable attention for the treatment of ALI, due to significant therapeutic effects and relatively low toxicity. These therapeutic products act as antioxidant and anti-inflammatory agents to arrest the key pathways responsible for progression of ALI.Ginger (Zingiber officinale Roscoe) has been widely used throughout the world because of its aromatic spicy nature. In Chinese and Indian traditional medicine in particular it has also been used as a herbal therapy, with benefits for treating a wide range of ailments including tenderness, heartburn, queasiness, vomiting, aches/pain, the common cold and diarrhea. Compounds derived from phenylpropanoids act as a basic skeleton for the active-constituents of ginger, including gingerols and shogaols. Both of these products are present in fresh and dried ginger. Gingerol, or sometimes referred to as [6]-gingerol, is the active constituent of fresh ginger. In contrast, shogaol (or 6-shogaol), a dehydrated product of gingerols, is found in larger amounts in dried as opposed to fresh ginger. The results of previous studies suggest that shogaol has prominent anti-proliferative and anti-inflammatory effects. With respect to its anti-inflammatory action,6-shogaol suppressed the generation of metabolites of both cyclooxygenase and lipoxygenase, and arachidonic acid. Moreover, the organic extracts from dried ginger rhizomes inhibited lipopolysaccharide (LPS)-induced prostaglandin E2 (PGE2) production. However, there have been no or very limited studies to elucidate the effect of 6-shogaol on LPS-stimulated ALI. Based on these considerations, the present study evaluated the pharmacological effects of 6-shogaol (6-SH) in an LPS-induced mouse model of ALI, and its mechanism of action.In recent years, through the computer software to assist drug design method, with its advantages of fast, economical and efficient in drug research and development plays an increasingly important role, among them, the molecular docking technology as the key technology of computer aided drug design, has become an important method in drug research and development, and become the frontier of research at home and abroad.The principle of molecular docking is through small molecule biological macromolecular ligand and the receptor interaction, predict the combination mode and affinity, and thus achieve a kind of important method based on the structure of the drug design. It can study the detailed interaction between drug molecules and their targets, can also be used to discover and optimization of drug leads.With the rapid development of computer software, easy to use protein simulations and drug design and optimization tools came into being.Through high quality function of the graphical interface, the science of computing and integrated digital environment, make the protein structure and function of the micro presents in sight. On the whole, this method can consider the combination of ligand and receptor effect, better to avoid the local effect is better, the overall situation of the combination of poor, and gene, protein expression methods complement each other, complement each other.Therefore, this topic proposed by copying sepsis lung injury in mice model, from five aspects:alveolar capillary permeability, expression of inflammatory cytokines, lung tissue pathology manifestations, expression of NF-kappa B and 6-SH docking with the NF-kappa B,to discusses the protective effect and mechanism of 6-shogaol inALI.Part I Protective effects of 6-shogaol on membrane permeability of alveolar capillaries in mice with acute lung injury induced by lipopolysaccharideObjectiveTo intisvetigate effects of dexmedetomidine-ulinastatin combination on acute lung injury induced by Lipopolysaccharide in rats.Material and methodsIn order to elucidate the effect of 6-shogaol, it was procured from the Sigma Aldrich. Whereas, the Dexamethasone (DEX) was supplied by Changle Pharmaceutical Co. (China).The TNF-a, IL-6 and IL-1β ELISA kits were procured from Biolegend. The assay kit obtained from Nanjing Jiancheng Bioengineering Institute of Nanjing (China) was used for the estimation of myeloperoxidase (MPO).Statistical analysisThe experimental data are presented as mean±S.E.M. Whereas, ANOVA followed by Dunnett’s test was used to compare between experimental groups. P-values of 0.05 or less was considered statistically significant. The analysis was performed using SPSS 17.0.AnimalsThe Male BALB/c mice, weighing approximately 18 to 20 g, were utilized for performing biological assays. All animals were housed in appropriate laboratory cages and fed with standard laboratory diet and water ad libitum. Before starting any experiment, all mice were acclimatized to the standard laboratory environment for a minimum of 3 days Whereas, animal experiments were performed in accordance with an Institutional Animal Ethical Committee.Induction of ALI in mice via administration of LPSSix groups of mice were performed taking 12 healthy male BALB/c mice in each group. The classification of the group are as follows:one group serves as a control group, second group only LPS treated; while third, fourth and fifth groups receives LPS+6-SH at a concentration of 10,20 and 40 mg/kg, respectively. The last group contains LPS and DEX. The 6-SH and DEX were administered intra-peritoneally. While the mice of control and LPS group received an equal volume of NS instead of a drug. After 1 h, the mice were made unconscious with an inhalation of diethyl ether to induce lung injury via intranasal instillation of 10 μg of NS in 50 μl. The control mice were given 50μl NS without inducing agent. The bronchoalveolar lavage fluid (BALF) was collected three times through a tracheal cannula with sterile NS upto a total volume of 1.3 ml.Lung wet to dry weight (W/D) ratio measurement and protein analysisThe lungs were removed after the mice were euthanized, and the corresponding wet weight was quantified. Thus, to obtain dry weight, the lungs were placed in a drying oven at 60℃ for 24 h. The relative difference of the wet lung against the dry lung was easily interpreted as a severity of edema in the tissues. The BALF was collected and protein content was determined by BCA method. The protein level was expressed in milligram protein per millilitre in BALF.Inflammatory cell counts of BALFTo quantify the inflammatory cell count, pellets of the cells were obtained from centrifugation of fluid collected from each sample. The centrifugation was carried out at 4℃,3000 rpm (800xg) for 10 min. The resulting pellets were re-suspended in PBS for total cell counts using a haemocytometer.ResultsEffect of 6-SH on LPS-induced lung W/D ratio and protein concentration in the BALFIn acute lung injury, the lungs are associated with pulmonary edema. Therefore, the effect of 6-SH was determined on the W/D ratio of the lungs. The ratio has been determined after seven hours of instillation of LPS via the intranasal route. After that, the lung W/D ratio and total protein concentration was calculated. The results showed that, the lung W/D ratio was augmented after LPS administration in comparison of the control group (P<0.01). However, the pre-treatment with 6-SH at different concentrations, viz.,10,20 and 40 mg/kg or DEX drastically reduces the lung W/D ratio compared with the LPS group (P<0.05). The total protein concentration of BALF was found significantly elevated in the case of LPS group. However, in comparison with the pre-treatment of LPS group, the pre-treatment of 6-SH (10,20 and 40 mg/kg) or DEX diminishes the total protein concentration significantly (P<0.05 or P<0.01).Effect of 6-SH on the inflammatory cell count in the BALF of LPS-induced ALI miceThe lung injury becomes worse after generation of pro-inflammatory cytokines, which released as a result of the early inflammatory response. These cytokines, including TNF-α, IL-1β and IL-6 and other inflammatory mediators played a critical role in progression of ALI in its severe form. It has been found that patients affected by ARDS showed elevated level of TNF-α, IL-1β and IL-6 in the BALF. Apart from its activity to intensify the inflammatory process to cause associated injury, these cytokines also recruit neutrophils into the lung which results in higher MPO activity. This higher activity leads to the release of granular enzymes, which could be associated with lung injury and pulmonary edema. Therefore, the number of inflammatory cells, such as neutrophils and macrophages, in BALF were analyzed at 7 h after LPS challenge.the number of total cells, neutrophils and macrophages compared with that of the control group (P<0.01) was found elevated after administration of LPS. Nonetheless, pre-treatment with 6-SH (10,20 and 40 mg/kg) or DEX appreciably lessen the number of total cells (P<0.01), neutrophils (P<0.01), and macrophages (P<0.01). These results indicated that,6-SH exert protective effects in ALI of mice.Conclusions6-Shogaol significantly inhibits the elevated level of the influx of neutrophil, protein concentration and edema.Part II Effects of 6-shogaol on inflammatory cytokines in BALF of lipopolysaccharide-induced acute lung injury in miceObjectiveTo intisvetigate effects of 6-shogaol on inflammatory cytokines in BALF of lipopolysaccharide-induced acute lung injury in miceMaterial and methodsIn order to elucidate the effect of 6-shogaol, it was procured from the Sigma Aldrich. Whereas, the Dexamethasone (DEX) was supplied by Changle Pharmaceutical Co. (China).The TNF-a, IL-6 and IL-1β ELISA kits were procured from Biolegend. The assay kit obtained from Nanjing Jiancheng Bioengineering Institute of Nanjing (China) was used for the estimation of myeloperoxidase (MPO).Statistical analysisThe experimental data are presented as mean ±S.E.M. Whereas, ANOVA followed by Dunnett’s test was used to compare between experimental groups. P-values of 0.05 or less was considered statistically significant. The analysis was performed using SPSS 17.0.AnimalsThe Male BALB/c mice, weighing approximately 18 to 20 g, were utilized for performing biological assays. All animals were housed in appropriate laboratory cages and fed with standard laboratory diet and water ad libitum. Before starting any experiment, all mice were acclimatized to the standard laboratory environment for a minimum of 3 days Whereas, animal experiments were performed in accordance with an Institutional Animal Ethical Committee.Induction of ALI in mice via administration of LPSSix groups of mice were performed taking 12 healthy male BALB/c mice in each group. The classification of the group are as follows:one group serves as a control group, second group only LPS treated; while third, fourth and fifth groups receives LPS+6-SH at a concentration of 10,20 and 40 mg/kg, respectively. The last group contains LPS and DEX. The 6-SH and DEX were administered intra-peritoneally. While the mice of control and LPS group received an equal volume of NS instead of a drug. After 1 h, the mice were made unconscious with an inhalation of diethyl ether to induce lung injury via intranasal instillation of 10 μg of LPS in 50 μl. The control mice were given 50 μl PBS without inducing agent. The bronchoalveolar lavage fluid (BALF) was collected three times through a tracheal cannula with sterile NS upto a total volume of 1.3 ml.Cytokine assaysThe concentrations of various pro-inflammatory markers, such as, TNF-a, IL-6, and IL-1β in the BALF were measured by ELISA kits according to the standard protocol provided by the manufacture (BioLegend, Inc.). In a brief, the standards along with samples were added to the 96-well plate after treating the well-plate with specific monoclonal antibody. Further, a biotinylated detection antibody is added to the above, followed by the addition of avidin-horseradish peroxidase enzyme. The TMB substrate solution is subsequently added, which impart blue colour to the resulting solution. Finally, when the reaction was stopped, it changes the colour from blue to yellow. Thus, the optical density (OD) of the microplate was read at 450 nm.ResultEffect of 6-SH on generation of cytokines in the BALF of LPS-treated ALI miceAs discussed above, for the progression of ALI, the release of various pro-inflammatory cytokines have played a major role. Thus, the inhibition of the generation of these cytokines results in the loss of inflammatory response associated with it. Consequently,6-SH was analyzed after 6h of administration of LPS by ELISA for its activity against TNF-α, IL-1β and IL-6. in the influence of LPS, the levels of TNF-α, IL-6, and IL-1β in BALF were appreciably augmented in comparison to the control. However, these levels were reduced drastically after the introduction of 6-SH in different concentrations. It was surprising to note that, these effects were dependent upon the concentration of 6-SH.Conclusion6-Shogaol significantly inhibits the elevated level of various pro-inflammatory cytokines, e.g., TNF-α, IL-1β and IL-6.Part III Effects of 6-shogaol on Pulmonary MPO activity and Histopathological analysis of the lung tissue in mice withlipopolysaccharide-induced acute lung injuryObjectiveTo intisvetigate effects of 6-shogaol on Pulmonary MPO activity and Histopathological analysis of the lung tissue in mice withlipopolysaccharide-induced acute lung injuryMaterial and methodsIn order to elucidate the effect of 6-shogaol, it was procured from the Sigma Aldrich. Whereas, the Dexamethasone (DEX) was supplied by Changle Pharmaceutical Co. (China).The TNF-a, IL-6 and IL-1β ELISA kits were procured from Biolegend. The assay kit obtained from Nanjing Jiancheng Bioengineering Institute of Nanjing (China) was used for the estimation of myeloperoxidase (MPO).Statistical analysisThe experimental data are presented as mean±S.E.M. Whereas, ANOVA followed by Dunnett’s test was used to compare between experimental groups. P-values of 0.05 or less was considered statistically significant. The analysis was performed using SPSS 17.0.The Male BALB/c mice, weighing approximately 18 to 20 g, were utilized for performing biological assays. All animals were housed in appropriate laboratory cages and fed with standard laboratory diet and water ad libitum. Before starting any experiment, all mice were acclimatized to the standard laboratory environment for a minimum of 3 days Whereas, animal experiments were performed in accordance with an Institutional Animal Ethical Committee.Induction of ALI in mice via administration of LPSSix groups of mice were performed taking 12 healthy male BALB/c mice in each group. The classification of the group are as follows:one group serves as a control group, second group only LPS treated; while third, fourth and fifth groups receives LPS+6-SH at a concentration of 10,20 and 40 mg/kg, respectively. The last group contains LPS and DEX. The 6-SH and DEX were administered intra-peritoneally. While the mice of control and LPS group received an equal volume of NS instead of a drug. After 1 h, the mice were made unconscious with an inhalation of diethyl ether to induce lung injury via intranasal instillation of 10 μg of LPS in 50 μl. The control mice were given 50 μl NS without inducing agent. The bronchoalveolar lavage fluid (BALF) was collected three times through a tracheal cannula with sterile NS upto a total volume of 1.3 ml.Pulmonary MPO activity in acute lung injury miceMice were sacrificed 6h after LPS challenge. Lung tissues were frozen in liquid nitrogen and then homogenized in PBS. The MPO activities in the lung homogenates were examined by using a MPO determination kit (Nanjing JianCheng Bioengineering Institute, China). The rest homogenate was centrifuged at 2000xg for 10min at 4℃.Histopathological analysis of the lung tissueThe rest of the mice that were not used after BALF collection were utilized for histopathological examination. The lungs of these mice were sliced after fixing with 10% buffered formalin, embedded in paraffin. After staining with hematoxylin and eosin (H&E), the alterations in pathology of the lung tissues were observed under a normal light microscope at 200x magnification.ResultEffects of 6-SH on MPO activityMPO activity serves as an important marker of neutrophil influx in the tissue component. By measuring this activity, we could easily translate the accrual of the neutrophils in the pulmonary tissues. Thus, the LPS induced model of ALI showed the infiltration of neutrophils in the lungs; resulting in higher MPO activity. This excreted MPO by neutrophils results in the generation of MPO derived oxidant which ultimately leads to the tissue damage. Thus, inhibition of MPO would prevent the associated degradation of pulmonary tissue, the activity of MPO in tissues was significantly increased in comparison to the control group (P<0.01). However, administration of6-SH showed reduction in MPO activity in a dose dependent manner (10,20, and 40 mg/kg) (P<0.01) or DEX (P<0.01). This result indicates the protective effect of 6-SH on the MPO induced tissue damage.Effect of 6-SH on the histopathology of lungs of LPS-treated miceThe lungs of mice were harvested for 6h after challenging with LPS and were subjected HE staining, lung tissues from the control group showed a normal structure and no major histopathological changes were observed. Whereas, in the LPS challenged group, tissues showed marked inflammation due to infiltration of neutrophils along with local fibrosis. Additionally, it also showed the thickening of the alveolar wall and congestion of the pulmonary. Nevertheless, these pathological alterations were extensively reversed to normal upon treatment with 6-SH (10,20, and 40 mg/kg) and DEX (5 mg/kg) treatment.ConclusionThe effect of 6-Shogaol confirmed by histopathological examination of lung tissues and the activity of MPO,which suggest that insignificantly improve the pathological condition to normal in dose-dependent manner.Part Ⅳ Effects of 6-shogaol on the way of NF-κB in the lung tissue in mice with lipopolysaccharide-induced acute lung injuryObjectiveTo intisvetigate effects of 6-shogaol on NF-κB in the lung tissue in mice with lipopolysaccharide-induced acute lung injuryMaterial and methodsIn order to elucidate the effect of 6-shogaol, it was procured from the Sigma Aldrich. Whereas, the Dexamethasone (DEX) was supplied by Changle Pharmaceutical Co. (China).The concentrations of NF-κB were measured by western-blotting.Statistical analysisThe experimental data are presented as mean±S.E.M. Whereas, ANOVA followed by Dunnett’s test was used to compare between experimental groups. P-values of 0.05 or less was considered statistically significant. The analysis was performed using SPSS17.0.The Male BALB/c mice, weighing approximately 18 to 20 g, were utilized for performing biological assays. All animals were housed in appropriate laboratory cages and fed with standard laboratory diet and water ad libitum. Before starting any experiment, all mice were acclimatized to the standard laboratory environment for a minimum of 3 days Whereas, animal experiments were performed in accordance with an Institutional Animal Ethical Committee.Induction of ALI in mice via administration of LPSSix groups of mice were performed taking 12 healthy male BALB/c mice in each group. The classification of the group are as follows:one group serves as a control group, second group only LPS treated; while third, fourth and fifth groups receives LPS+6-SH at a concentration of 10,20 and 40 mg/kg, respectively. The last group contains LPS and DEX. The 6-SH and DEX were administered intra-peritoneally. While the mice of control and LPS group received an equal volume of NS instead of a drug. After 1 h, the mice were made unconscious with an inhalation of diethyl ether to induce lung injury via intranasal instillation of 10 μg of LPS in 50 μl. The control mice were given 50 μl NS without inducing agent.NF-κB activity in acute lung injury miceMice were sacrificed 6h after LPS challenge. Lung tissues were frozen in liquid nitrogen and then homogenized in PBS. The NF-κB activities in the lung homogenates were examined by western-blotting.ResultEffects of 6-SH on NF-κB activityNF-κB activity serves as a key marker of inflammation. Thus, inhibition of NF-κB would prevent the associated degradation of pulmonary tissue, the activity of NF-κB in tissues was significantly increased in comparison to the control group (P<0.01). However, administration of 6-SH showed reduction in NF-κB activity in a dose dependent manner (10,20, and 40 mg/kg) (P<0.01) or DEX (P<0.01). This result indicates the protective effect of 6-SH on the way of NF-κB induced tissue damage.ConclusionThe effect of 6-Shogaol confirmed by examination to the activity of NF-κB in lung tissues,which suggest that it significantly reduce the level of NF-κB in dose-depend ent manner.part V Analysis of docking between NF-κB and 6-shogaolObjectiveTo investigate effects between 6-shogaol and NF-κB with dockingMaterial and methodsProtein preparationDocking was carried out against structure of NF-kappa B p50 homodimer bound to a kappa B site for possible inhibitory effect of 6-SH. The co-crystallized ligands, bound water molecules and cofactors were separated from the proteins before starting any docking experiments. The CHARMm force field was used for the minimization of the target protein using steepest descent (gradient< 0.1) and conjugate gradient algorithms (gradient< 0.01). A suitable active-site was identified in the target protein via define active site module within the Discovery Studio 2.5.Ligand Fit dockingTo exemplify the molecular interactions of 6-SH necessary for inhibition of key enzymes, Ligand Fit, a shape-based method inbuilt in Discovery Studio 2.5 was utilized for docking analysis. It works on cavity detection algorithm, where, a shape comparison filter was combined with Monte Carlo technique to produce various ligand conformations. The most stable conformation of ligand was then allowed to dock into the active-site of a protein using the grid resolution of 0.5 A (default). During docking, the 5 A from the core of the binding site was kept flexible during the minimization. This was carried out to calculate the non-bonded interactions between the ligand and catalytic residues of the target protein. The resultant docked pose was selected for further analysis and image generation.ResultEffect of 6-SH on the NF-κBThe diverse cellular process could be easily controlled by transcription factor NF-κB. In dormant condition, NF-κB will exist in bound form with its inhibitor (IκBs) in cytoplasm. Once it is triggered to its active form, the p65units of NF-κB dissociates from its inhibitory protein IκB-α and reaches to the nucleus. After arriving in the nucleus, it attaches to κB binding sites and activate the transcription of specific target genes such as TNF-α, IL-1β and IL-6. Since we have explored the initial effects of 6-SH on the level of these cytokines, then, it is imperative to examine the effect 6-SH on the inhibition of NF-κB.For this, a molecular docking analysis of 6-SH has been undertaken with the NF-κB protein model. LigandFit within Discovery Studio 2.5 was used to carry out this experiment as per the standard protocol as mentioned in the experimental section. Results showed that,6-SH create excellent H-bonds with residues like, Lys145 and Lys146, fig.7. It also showed the formation of the one pi-cation interaction with Lys145. It is surprising the note that, 6-SH was efficiently buried in the active site of the NF-κB, situated between Chain A and Chain B. Docking analysis showed that,6-SH was located deep in the cleft lined with Tyr57, Val58 and Cys59, Tyr143, Lys145 and Lys146 of the Chain B of p50 NF-κB. In terms of scoring profile,6-SH was again revealed as efficient ligand to control the activity of NF-κB with DOCK SCORE of 41.27 and ligand internal energy of -3.52.ConclusionIt is proved by dockingthe effect 6-SH on the inhibition of NF-κB. |
PDF Full Text Request