Effect of the protein kinase RNA-like endoplasmic reticulum kinase pathway in endoplasmic reticulum stress on hepatic stellate cell activation and collagen type I expression
Objective To investigate the effect of the protein kinase RNA-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α) pathway in endoplasmic reticulum stress on the activation of hepatic stellate cells (HSC).Methods Pathological sections of normal liver tissue after surgery were collected from 11 patients with hepatic fibrosis (S1-S4) and 9 patients with hepatic hemangioma and hepatic adenoma confirmed by liver biopsy, and immunohistochemistry was used to measure the protein expression levels of PERK, eIF2α, and C/EBP homologous protein (CHOP). Human HSC-LX2 cells were treated with different concentrations of the endoplasmic reticulum stress inducer thapsigargin (0, 125, 250, 500, and 1 000 nmol/L), and qRT-PCR was used to measure the mRNA expression level of PERK, while Western blot was used to measure the protein expression levels of PERK, inositol requiring protein 1 (IRE1), activating transcription factor 6 (ATF6), CHOP, and α-smooth muscle actin (α-SMA). The method of lentivirus transfection was used to construct a PERK stable overexpression LX-2 group and a control group; qRT-PCR was used to measure the mRNA expression levels of PERK, eIF2α, and α-SMA, Western blot was used to measure the protein expression levels of PERK, phosphorylated eIF2α (p-eIF2α), and α-SMA, and immunofluorescence assay was used to measure the expression of collagen type I alpha 1 (COL1A1). The independent samples t-test was used for comparison of normally distributed continuous data between two groups; a one-way analysis of variance was used for comparison between multiple groups, and the least significant difference t-test was used for further comparison between two groups. The Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups.Results Compared with normal liver tissue, the liver tissue of patients with hepatic fibrosis had significantly higher expression levels of PERK, eIF2α, and CHOP (Z=-3.56, t=-5.75, Z=-3.52, all P<0.001). Compared with the solvent group, the groups treated with different concentrations of thapsigargin had significant increases in the expression levels of the endoplasmic reticulum-associated proteins PERK, CHOP, IRE1, ATF6, and α-SMA (all P<0.05). Compared with the control group, the PERK stable overexpression group had significant increases in the mRNA expression levels of PERK, eIF2α, and α-SMA and the protein expression levels of PERK, p-eIF2α, and α-SMA (all P<0.05), and immunofluorescence assay showed a significant increase in the expression level of COL1A1 in the PERK stable overexpression group (P<0.05).Conclusion PERK overexpression can induce the expression of α-SMA and COL1A1 in LX-2 cells, suggesting that the PERK signaling pathway might be one of the important mechanisms of HSC activation.
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Unidimensional statistical analysis of intestinal differential metabolices before and after treatment with Yiqi Yangyin Jiedu Huayu prescription
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Multidimensional statistical analysis of intestinal differential metabolites before and after treatment with Yiqi Yangyin Jiedu Huayu prescription
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