Role Of Glycolytic Reprogramming In Silicotic Fibrosis And Its Targeted Intervention Mechanism

Objectives To study the mechanism of glycolytic reprogramming in regulating the occurrence and development of silicotic fibrosis.The effect and mechanism of inhibiting glycolytic pathway to alleviate silicotic fibrosis were investigated by using N-acetyl-seryl-aspartyl-lysyl-proline（Ac-SDKP）and Oxamate to block glycolytic reprogramming.Methods The autopsy specimens of coal workers’pneumoconiosis and the serum of pneumoconiosis patients were selected as the research objects.Silicosis model of rats was established by dynamic silica dust exposure control system.Silicosis model of mice was established by intratracheal instillation of silica suspension.The silicotic rats were treated with Ac-SDKP intervention.Silicotic mice were given 100 mg/kg and 500 mg/kg Oxamate intervention treatment.NR8383 alveolar macrophages were induced by Si O₂,and treated with si RNA-Ldha,Ac-SDKP,Lactate and Oxamate,respectively.HE and Sirius red staining were used to observe the pathological changes and collagen deposition in lung tissues of patients with coal workers’pneumoconiosis,rats and mice.Immunohistochemistry and immunofluorescence staining were used to observe the expression of LDHA and its co-expression with CD68 in lung tissues,and the co-expression of monocyte chemoattractant protein-1（MCP1）and LDHA,arginase-1（Arg-1）and LDHA in NR8383 cells.Western blotting was used to detect the protein expression of collagen type I（Col I）,α-smooth muscle actin（α-SMA）,hexokinase2（HK2）,pyruvate kinase M2（PKM2）,and LDHA in animal model lung tissues and NR8383 cells.Western blotting was used to detect the protein expression of inducible nitric oxide synthase（i NOS）,tumor necrosis factor-α（TNF-α）,Arg-1,interleukin-10（IL-10）and MCP1 in NR8383 cells.The m RNA expressions of Hk2,Pkm and Ldha in lung tissues of animal models were detected by real-time fluorescence quantitative PCR.The expressions of phospho-protein kinase RNA-like endoplasmic reticulum kinase（p-PERK）,phospho-inositol-requiring kinase 1α（p-IRE-1α）and phospho-eukaryotic initiation factor 2α（p-e IF-2α）in mice lung tissues and/or NR8383 cells were detected by immunohistochemical/cytochemical staining and immunofluorescence staining and/or western blot.LDH enzyme activity in serum of patients with pneumoconiosis and rats were detected by LDH colorimetry.The concentration of lactate in rat lung tissue and NR8383 cell medium was detected by lactate colorimetry.The level of ROS in NR8383 cells was detected by DCFH-DA fluorescence probe.The energy metabolism of NR8383 cells was measured by Seahorse extracellular flux analyzer,including extracellular acidification rate（ECAR）and oxygen consumption rate（OCR）.Results 1 The typical pathologic evolution of coal（silicotic）pulmonary fibrosis was observed in autopsy lung tissue of coal workers’pneumoconiosis,including macrophage alveolitis,cellular nodules,cellular fibrous nodules and fibrous（hyaline）nodules.Dust spot（focal）emphysema and dusty diffuse interstitial fibrosis were also seen.Typical cellular and cellular fibrous nodules can be seen in the lungs of silicotic rats and mice,and the nodules contain fibrous strip collagen fibers.2 Based on high-throughput transcriptome sequencing,the m RNA expression profile of key glycolysis enzymes was mapped based on the screening of m RNAs expression results related to silicosis in rats.It was found that some key glycolysis enzymes,such as Hk2,Gapdh,Pgk1,Pgam1,Eno1 and Ldha,were up-regulated in lung tissues of silicotic rats.3 LDHA was strongly expressed in macrophages of coal workers’pneumoconiosis and in silicotic nodules of silicotic rats and mice.In coal workers’pneumoconiosis,the area of macrophage alveolar inflammation is particularly obvious.In rat and mouse silicosis models,it is mainly located in silicotic nodules,and LDHA and CD68are co-expression in macrophages in the lesion area.Compared with the control group,the protein and m RNA expressions of HK2,PKM2 and LDHA,as well as the protein expressions of Col I andα-SMA,were up-regulated in the silicosis model group of rats and mice.In the silicosis model group of mice,p-PERK and p-IRE-1αwere positively expressed,and the protein levels of Col I,LDHA,p-PERK,p-IRE-1αand p-e IF-2αwere up-regulated.LDH enzyme activity was increased in serum of patients with coal workers’pneumoconiosis（stage I,II and III）and silicotic rats.4 In Si O₂-induced NR8383 alveolar macrophages,the expression of key enzymes of glycolysis and activation related factors of macrophages were enhanced,and the content of lactate was increased in a dose-dependent manner.In addition,the positive expression of p-PERK,p-IRE-1αand ROS increased with the increase of Si O₂or lactate induced concentration,and the protein expression of p-PERK,p-IRE-1αand p-e IF-2αalso increased in a dose-dependent manner.Ldha silencing can inhibit macrophage activation,reduce lactate content and ROS generation.These results suggest that glycolytic reprogramming is involved in the process of silicotic fibrosis and promotes the formation and progression of silicosis.5 In the silicotic rat model,after Ac-SDKP intervention,the fluorescence expression of LDHA in silicotic nodule was significantly weakened,the lactate content in lung tissue was significantly reduced,and the LDH enzyme activity in serum was reduced.Meanwhile,the expression levels of Col I,α-SMA,key enzymes of glycolysis and macrophage activation related factors were significantly decreased.In the macrophage culture model,Ac-SDKP treatment significantly reduced the co-expression of LDHA/MCP1 and LDHA/Arg-1 induced by Si O₂,and inhibited the expression of macrophage activation related factors,reduced lactate content and ROS generation.It is suggested that Ac-SDKP can antagonize silicotic fibrosis by targeting macrophage glycolytic reprogramming.6 Compared with the silicosis model group,100 mg/kg and 500 mg/kg Oxamate could reduce the number,area and collagen deposition of silicotic nodules in the lung of silicotic mice,and decrease the protein expression of Col I,LDHA,p-PERK and p-IRE-1αin the lung tissue of silicotic mice.In the macrophage culture model,the intervention of Oxamate could reduce p-PERK,p-IRE-1αand ROS positive expression mediated by Si O₂or lactate,as well as high level ECAR and lactate content,and increased low level OCR.It is suggested that Oxamate can play an anti-fibrosis role by blocking glycolytic metabolism,inhibiting macrophage activation and endoplasmic reticulum stress（ERS）.Conclusions 1 Abnormal activation of glycolytic metabolism is accompanied by the process of silicotic fibrosis.2 Ac-SDKP antagonizes silicotic fibrosis by inhibiting Si O₂-mediated glycolysis metabolism enhancement and macrophage activation.3 Oxamate can inhibit the enhancement of macrophage glycolysis and ERS mediated by Si O₂ through targeted intervention of LDHA,thereby inhibiting the occurrence and progress of silicotic fibrosis.Figure 70;Table 5;Reference 285...