Background:Hepatic fibrosis is a common chronic liver disease in clinic,the purpose of our study is to explore potential biomarkers to provide a theoretical basis for the treatment of liver fibrosis with pirfenidone.M...Background:Hepatic fibrosis is a common chronic liver disease in clinic,the purpose of our study is to explore potential biomarkers to provide a theoretical basis for the treatment of liver fibrosis with pirfenidone.Methods:We downloaded a gene-sequencing dataset and a single-cell dataset from the GEO database and pirfenidone target genes from three different databases.First,we performed GO,KEGG,and DO analysis on pirfenidone target genes.Then,we grouped the liver tissue sequencing data(GSE162694)in the sequencing data set(N-F0 group and F1-F4 group)and performed gene expression differential analysis on these two groups,weighted gene co-expression network analysis and gene Enrichment analysis.Finally,we intersected the significantly upregulated genes in the F1-F4 group with the pirfenidone target genes and performed PPI network analysis.In order to further explore the expression of both pirfenidone drug target genes and liver fibrosis disease genes(PDLFG)in different immune cells of liver tissue,we used the CD45+cell data in the GSE136103 data set for further analysis.Results:A subnetwork consisting of CDC42,HNF4A,BHLHE40,CCDC71L,NR1H3,TNF,MGLL,GPT,SCD and PLIN1 was screened out,and by analysis,we finally identified the SCD as PDLFG.In single-cell sequencing analysis,we found that SCD was highly expressed in M2-polarized macrophages.Conclusion:SCD may be an important target protein to inhibit the progression of liver fibrosis.展开更多
AIM: To determine the effect of pirfenidone on the activated human Müller cells by platelet-derived growth factor-BB(PDGF-BB). METHODS: The primary human Müller cells were separated from retinal tissues and ...AIM: To determine the effect of pirfenidone on the activated human Müller cells by platelet-derived growth factor-BB(PDGF-BB). METHODS: The primary human Müller cells were separated from retinal tissues and established the pathogenic model by stimulated with PDGF-BB. The Müller cells behaviour of normal group and the model group was measured by MTT assay, Trypan blue assay, cell migration assay, and collagen contraction assay. The expression of transforming growth factor(TGF)-β1,-β2, and pigment epithelium-derived factor(PEDF) was estimated with realtime polymerase chain reaction(PCR), Western blot and immunofluorescence analyses. RESULTS: A pathogenic/proliferative model of Müller cells was established by stimulating normal cultured Müller cells with 10 ng/mL PDGF-BB for 48 h. After treated with 0.2 and 0.3 mg/mL pirfenidone, the proliferation, migration and collagen contraction was statistically significantly depressed in the model group compared with the normal groups. The expression levels of TGF-β1 and TGF-β2 were significantly down-regulated, while the PEDF expression was significantly up-regulated after treated with 0.2 and 0.3 mg/mL pirfenidone in the model group. CONCLUSION: Pirfenidone effectively suppress the proliferation, migration and collagen contraction of the human Müller cells stimulated with PDGF-BB through down-regulation of TGF-β1/TGF-β2 and up-regulation of PEDF.展开更多
A simple, rapid and rugged RP-HPLC method was developed for evaluation and quantification of impurities present in Pirfenidone (PFD) drug substance. Impurities were separated and determined on a Zorbax RX-C18 column (...A simple, rapid and rugged RP-HPLC method was developed for evaluation and quantification of impurities present in Pirfenidone (PFD) drug substance. Impurities were separated and determined on a Zorbax RX-C18 column (250 mm length, 4.6 mm inner diameter and 5.0 μm particle size, octadecylsilane chemically bonded to porous silica) with 0.02 M KH2PO4 buffer and acetonitrile as mobile phase using a simple gradientelution program. The column flow rate of 1.0 mL per minute was used for the separation. The detection wave length was fixed at 220 nm. The method was substantiated with respect to specificity, precision, linearity, range, accuracy, ruggedness, limit of detection and quantitation. The impurities were identified as 2-hydroxy-5-methylpyridine and Iodobenzene. The linearity range obtained was 0.017 to 0.380 μg/mL for 2-hydroxy-5-methylpyridine, 0.047 to 0.382 μg/mL for Pirfenidone and 0.030 to 0.99 μg/mL for Iodobenzene with the retention times of 3.248 min, 10.608 min and 24.241 min for 2-hydroxy-5-methylpyridine, Pirfenidone and Iodobenzene, respectively. The percentage recoveries of 2-hydroxy-5-methylpyridine and Iodobenzene were in the range of 94.08% - 104.12%. The LOD and LOQ values were found 0.000005 mg/mL, 0.000017 mg/mL for 2-hydroxy-5-methylpyridine and 0.009 μg/mL, 0.030 μg/mL for Iodobenzene, respectively. The method is found to be suitable for the quantitation of impurities along with Pirfenidone drug substance. The method was validated as per the International Conference on Harmonization (ICH) guidelines.展开更多
Objective To investigate the optimal dosage of pirfenidone for the treatment of pulmonary fibrosis induced by bleomycin in Wistar rats, and the alteration of expressions of transforming growth factor beta-1 (TGF-β_ 1...Objective To investigate the optimal dosage of pirfenidone for the treatment of pulmonary fibrosis induced by bleomycin in Wistar rats, and the alteration of expressions of transforming growth factor beta-1 (TGF-β_ 1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and matrix metalloproteinase-13 (MMP-13) in lung tissue. Methods Male Wistar rats were endotracheally instilled with bleomycin or normal saline. Pirfenidone (25-[KG*8]800 mg·kg -1·d -1), dexamethasone (3 mg/kg), or 1% carboxymethylcellulose sodium were given daily by feed 2 days before instillation of bleomycin. Groups T7 and T14 were fed pirfenidone 50 mg·kg -1·d -1 at 7 days or 14 days after bleomycin instillation. Lungs were harvested at 28 days after bleomycin instillation. Patholological changes in lung tissues were evaluated with HE staining. Lung collagen was stained by sirius red and measured by content of hydroxyproline. Expression of proteins of TGF-β_ 1, TIMP-1, and MMP-13 were detected by Western blotting. Results At doses of 25, 50, and 100 mg·kg -1·d -1, pirfenidone had significant anti-fibrotic effects for bleomycin-induced rat pulmonary fibrosis, and these effects were most significantly attenuated at the dosage of 50 mg·kg -1·d -1 (HE: P<0.01, P<0.01, and P=0.064; sirius red: P<0.05, P<0.01, and P<0.05; hydroxyproline: P=0.595, P<0.01, and P=0.976). Pirfenidone at a dosage of[KG*3]50 mg·kg -1·d -1 inhibited protein expression of TGF-β_ 1 and TIMP-1 in lung tissue in the early phase (0.79 and 0.75 times of control group), but had no effect on expression of MMP-13. Conclusion Low dose pirfenidone, especially at dosage of 50 mg·kg -1·d -1, has significant anti-fibrotic effects on bleomycin-induced rat pulmonary fibrosis. Pirfenidone partially inhibits the enhancement of the expression of TGF-β_ 1 and TIMP-1 in lung tissue.展开更多
Background:Keloids are benign fibroproliferative skin lesions that are difficult to treat and become a lifetime predicament for patients.Several treatment modalities have been put forth,but as yet no satisfactory appr...Background:Keloids are benign fibroproliferative skin lesions that are difficult to treat and become a lifetime predicament for patients.Several treatment modalities have been put forth,but as yet no satisfactory approach to the prevention or treatment of keloids has been identified.The process of epithelial-to-mesenchymal transition(EMT)has been implicated in keloid scarring,as keloid keratinocytes display an EMT-like phenotype.This study investigated the potential of pirfenidone,an antifibrotic agent,to counteract EMT-like alterations in keloid keratinocytes,including gene expression,cell migratory and proliferative functions.Methods:Normal and keloid keratinocytes were isolated from discarded normal skin tissues and from resected keloid tissues,respectively.Cells were quiesced for 24 h without epidermal growth factor DS-Qi1MCDigital and were exposed to transforming growth factor-beta1(TGF-β1;10 ng/mL),with or without pirfenidone(400μg/mL),for an additional 24 h.The effects of pirfenidone on cytotoxicity,cell migration,cell proliferation,and on expression of genes and proteins involved in EMT were assayed.Statistical significance was determined by two-way ANOVA using Sigma Plot.Results:We found that pirfenidone did not elicit any cytotoxic effect at concentrations up to 1000μg/mL.A statistically significant dose-dependent decrease in basal cell proliferation rate was noted in both normal and keloid keratinocytes when exposed to pirfenidone at concentrations ranging from 200 to 1000μg/mL.Pirfenidone significantly decreased basal cell migration in both normal and keloid keratinocytes,but a significant decrease in TGF-β1-induced cell migration was seen only in keloid keratinocytes.Significant inhibition of the expression of TGF-β1-induced core EMT genes,namely hyaluronan synthase 2,vimentin,cadherin-11,and wingless-type MMTV integration site family,member 5A along with fibronectin-1,was observed in both normal and keloid keratinocytes treated with pirfenidone.In addition,the protein levels of vimentin and fibronectin were significantly reduced by pirfenidone(400μg/mL)in both normal and keloid keratinocytes.Conclusions:For the first time,this study shows the efficacy of pirfenidone in inhibiting the EMTlike phenotype in keratinocytes derived from keloids,suggesting that pirfenidone may counteract a critical contributor of keloid progression and recurrence.展开更多
We repurposed the antifibrotic drug pirfenidone—which is approved for treatment of idiopathic lung fibrosis—in a series of patients with nonalcoholic steatohepatitis-related cirrhosis.Our report demonstrates the obs...We repurposed the antifibrotic drug pirfenidone—which is approved for treatment of idiopathic lung fibrosis—in a series of patients with nonalcoholic steatohepatitis-related cirrhosis.Our report demonstrates the observed improvements in necroinflammation and regression of cirrhosis with pirfeni-done use for 12-weeks,associated with classical hepatic repair complex features on follow-up liver biopsies.This novel work could help stimulate further randomized trials of pirfe-nidone in patients with nonalcoholic steatohepatitis-related liver fibrosis or cirrhosis,for whom no recommended drug treatments exists currently.展开更多
Renal tubulointerstitialfibrosis(TIF)is the common end stage of various chronic renal diseases,and pirfenidone(PFD)is a novel,broad-spectrum anti-fibrotic compound but little is known about its effect and mechanism of a...Renal tubulointerstitialfibrosis(TIF)is the common end stage of various chronic renal diseases,and pirfenidone(PFD)is a novel,broad-spectrum anti-fibrotic compound but little is known about its effect and mechanism of action on renal TIF.In this work,we employed a unilateral ureteral obstruction(UUO)rat model to investigate the apoptosis of renal tubular epithelial cells(RTC)after PFD treatment.Thirty-five Sprague Dawley(SD)rats were randomized into three groups:sham-operated group(n=7),UUO group(n=14)and PFD group(n=14).All rats were sacrificed at day 7 or 14 after operation.Renal histology was studied by using periodic acid schiff reagent(PAS)and Masson trichromic stain(MASSON);apoptosis was detected by in situ terminal deoxynucleotide transferase-mediated dUTP-biotin nick end-labeling(TUNEL)method;tubular caspase-3 expression was assessed by immunohistochemistry.The content of malondialdehyde(MDA)and total activity of superoxide dismutase(T-SOD)in the renal cortex were determined by chemical colorimetry method.TIF,apopto-sis of RTC,tubular expression of caspase-3 and the content of MDA were increased in the UUO group compared with those in the sham-operated group,and were ameliorated significantly by PFD treatment(P<0.05).The activity of SOD was decreased in the UUO group,but was increased by PFD treatment(P<0.05).Our results showed that PFD could ameliorate TIF in the UUO group,and the possible mechanism was by reducing the apoptosis of RTC,which involved oxidative stress and caspase-3.展开更多
文摘Background:Hepatic fibrosis is a common chronic liver disease in clinic,the purpose of our study is to explore potential biomarkers to provide a theoretical basis for the treatment of liver fibrosis with pirfenidone.Methods:We downloaded a gene-sequencing dataset and a single-cell dataset from the GEO database and pirfenidone target genes from three different databases.First,we performed GO,KEGG,and DO analysis on pirfenidone target genes.Then,we grouped the liver tissue sequencing data(GSE162694)in the sequencing data set(N-F0 group and F1-F4 group)and performed gene expression differential analysis on these two groups,weighted gene co-expression network analysis and gene Enrichment analysis.Finally,we intersected the significantly upregulated genes in the F1-F4 group with the pirfenidone target genes and performed PPI network analysis.In order to further explore the expression of both pirfenidone drug target genes and liver fibrosis disease genes(PDLFG)in different immune cells of liver tissue,we used the CD45+cell data in the GSE136103 data set for further analysis.Results:A subnetwork consisting of CDC42,HNF4A,BHLHE40,CCDC71L,NR1H3,TNF,MGLL,GPT,SCD and PLIN1 was screened out,and by analysis,we finally identified the SCD as PDLFG.In single-cell sequencing analysis,we found that SCD was highly expressed in M2-polarized macrophages.Conclusion:SCD may be an important target protein to inhibit the progression of liver fibrosis.
文摘AIM: To determine the effect of pirfenidone on the activated human Müller cells by platelet-derived growth factor-BB(PDGF-BB). METHODS: The primary human Müller cells were separated from retinal tissues and established the pathogenic model by stimulated with PDGF-BB. The Müller cells behaviour of normal group and the model group was measured by MTT assay, Trypan blue assay, cell migration assay, and collagen contraction assay. The expression of transforming growth factor(TGF)-β1,-β2, and pigment epithelium-derived factor(PEDF) was estimated with realtime polymerase chain reaction(PCR), Western blot and immunofluorescence analyses. RESULTS: A pathogenic/proliferative model of Müller cells was established by stimulating normal cultured Müller cells with 10 ng/mL PDGF-BB for 48 h. After treated with 0.2 and 0.3 mg/mL pirfenidone, the proliferation, migration and collagen contraction was statistically significantly depressed in the model group compared with the normal groups. The expression levels of TGF-β1 and TGF-β2 were significantly down-regulated, while the PEDF expression was significantly up-regulated after treated with 0.2 and 0.3 mg/mL pirfenidone in the model group. CONCLUSION: Pirfenidone effectively suppress the proliferation, migration and collagen contraction of the human Müller cells stimulated with PDGF-BB through down-regulation of TGF-β1/TGF-β2 and up-regulation of PEDF.
文摘A simple, rapid and rugged RP-HPLC method was developed for evaluation and quantification of impurities present in Pirfenidone (PFD) drug substance. Impurities were separated and determined on a Zorbax RX-C18 column (250 mm length, 4.6 mm inner diameter and 5.0 μm particle size, octadecylsilane chemically bonded to porous silica) with 0.02 M KH2PO4 buffer and acetonitrile as mobile phase using a simple gradientelution program. The column flow rate of 1.0 mL per minute was used for the separation. The detection wave length was fixed at 220 nm. The method was substantiated with respect to specificity, precision, linearity, range, accuracy, ruggedness, limit of detection and quantitation. The impurities were identified as 2-hydroxy-5-methylpyridine and Iodobenzene. The linearity range obtained was 0.017 to 0.380 μg/mL for 2-hydroxy-5-methylpyridine, 0.047 to 0.382 μg/mL for Pirfenidone and 0.030 to 0.99 μg/mL for Iodobenzene with the retention times of 3.248 min, 10.608 min and 24.241 min for 2-hydroxy-5-methylpyridine, Pirfenidone and Iodobenzene, respectively. The percentage recoveries of 2-hydroxy-5-methylpyridine and Iodobenzene were in the range of 94.08% - 104.12%. The LOD and LOQ values were found 0.000005 mg/mL, 0.000017 mg/mL for 2-hydroxy-5-methylpyridine and 0.009 μg/mL, 0.030 μg/mL for Iodobenzene, respectively. The method is found to be suitable for the quantitation of impurities along with Pirfenidone drug substance. The method was validated as per the International Conference on Harmonization (ICH) guidelines.
基金Supported by National Ministry of Education Doctor Foundation of China(20020023045)
文摘Objective To investigate the optimal dosage of pirfenidone for the treatment of pulmonary fibrosis induced by bleomycin in Wistar rats, and the alteration of expressions of transforming growth factor beta-1 (TGF-β_ 1), tissue inhibitor of metalloproteinase-1 (TIMP-1), and matrix metalloproteinase-13 (MMP-13) in lung tissue. Methods Male Wistar rats were endotracheally instilled with bleomycin or normal saline. Pirfenidone (25-[KG*8]800 mg·kg -1·d -1), dexamethasone (3 mg/kg), or 1% carboxymethylcellulose sodium were given daily by feed 2 days before instillation of bleomycin. Groups T7 and T14 were fed pirfenidone 50 mg·kg -1·d -1 at 7 days or 14 days after bleomycin instillation. Lungs were harvested at 28 days after bleomycin instillation. Patholological changes in lung tissues were evaluated with HE staining. Lung collagen was stained by sirius red and measured by content of hydroxyproline. Expression of proteins of TGF-β_ 1, TIMP-1, and MMP-13 were detected by Western blotting. Results At doses of 25, 50, and 100 mg·kg -1·d -1, pirfenidone had significant anti-fibrotic effects for bleomycin-induced rat pulmonary fibrosis, and these effects were most significantly attenuated at the dosage of 50 mg·kg -1·d -1 (HE: P<0.01, P<0.01, and P=0.064; sirius red: P<0.05, P<0.01, and P<0.05; hydroxyproline: P=0.595, P<0.01, and P=0.976). Pirfenidone at a dosage of[KG*3]50 mg·kg -1·d -1 inhibited protein expression of TGF-β_ 1 and TIMP-1 in lung tissue in the early phase (0.79 and 0.75 times of control group), but had no effect on expression of MMP-13. Conclusion Low dose pirfenidone, especially at dosage of 50 mg·kg -1·d -1, has significant anti-fibrotic effects on bleomycin-induced rat pulmonary fibrosis. Pirfenidone partially inhibits the enhancement of the expression of TGF-β_ 1 and TIMP-1 in lung tissue.
文摘Background:Keloids are benign fibroproliferative skin lesions that are difficult to treat and become a lifetime predicament for patients.Several treatment modalities have been put forth,but as yet no satisfactory approach to the prevention or treatment of keloids has been identified.The process of epithelial-to-mesenchymal transition(EMT)has been implicated in keloid scarring,as keloid keratinocytes display an EMT-like phenotype.This study investigated the potential of pirfenidone,an antifibrotic agent,to counteract EMT-like alterations in keloid keratinocytes,including gene expression,cell migratory and proliferative functions.Methods:Normal and keloid keratinocytes were isolated from discarded normal skin tissues and from resected keloid tissues,respectively.Cells were quiesced for 24 h without epidermal growth factor DS-Qi1MCDigital and were exposed to transforming growth factor-beta1(TGF-β1;10 ng/mL),with or without pirfenidone(400μg/mL),for an additional 24 h.The effects of pirfenidone on cytotoxicity,cell migration,cell proliferation,and on expression of genes and proteins involved in EMT were assayed.Statistical significance was determined by two-way ANOVA using Sigma Plot.Results:We found that pirfenidone did not elicit any cytotoxic effect at concentrations up to 1000μg/mL.A statistically significant dose-dependent decrease in basal cell proliferation rate was noted in both normal and keloid keratinocytes when exposed to pirfenidone at concentrations ranging from 200 to 1000μg/mL.Pirfenidone significantly decreased basal cell migration in both normal and keloid keratinocytes,but a significant decrease in TGF-β1-induced cell migration was seen only in keloid keratinocytes.Significant inhibition of the expression of TGF-β1-induced core EMT genes,namely hyaluronan synthase 2,vimentin,cadherin-11,and wingless-type MMTV integration site family,member 5A along with fibronectin-1,was observed in both normal and keloid keratinocytes treated with pirfenidone.In addition,the protein levels of vimentin and fibronectin were significantly reduced by pirfenidone(400μg/mL)in both normal and keloid keratinocytes.Conclusions:For the first time,this study shows the efficacy of pirfenidone in inhibiting the EMTlike phenotype in keratinocytes derived from keloids,suggesting that pirfenidone may counteract a critical contributor of keloid progression and recurrence.
文摘We repurposed the antifibrotic drug pirfenidone—which is approved for treatment of idiopathic lung fibrosis—in a series of patients with nonalcoholic steatohepatitis-related cirrhosis.Our report demonstrates the observed improvements in necroinflammation and regression of cirrhosis with pirfeni-done use for 12-weeks,associated with classical hepatic repair complex features on follow-up liver biopsies.This novel work could help stimulate further randomized trials of pirfe-nidone in patients with nonalcoholic steatohepatitis-related liver fibrosis or cirrhosis,for whom no recommended drug treatments exists currently.
文摘Renal tubulointerstitialfibrosis(TIF)is the common end stage of various chronic renal diseases,and pirfenidone(PFD)is a novel,broad-spectrum anti-fibrotic compound but little is known about its effect and mechanism of action on renal TIF.In this work,we employed a unilateral ureteral obstruction(UUO)rat model to investigate the apoptosis of renal tubular epithelial cells(RTC)after PFD treatment.Thirty-five Sprague Dawley(SD)rats were randomized into three groups:sham-operated group(n=7),UUO group(n=14)and PFD group(n=14).All rats were sacrificed at day 7 or 14 after operation.Renal histology was studied by using periodic acid schiff reagent(PAS)and Masson trichromic stain(MASSON);apoptosis was detected by in situ terminal deoxynucleotide transferase-mediated dUTP-biotin nick end-labeling(TUNEL)method;tubular caspase-3 expression was assessed by immunohistochemistry.The content of malondialdehyde(MDA)and total activity of superoxide dismutase(T-SOD)in the renal cortex were determined by chemical colorimetry method.TIF,apopto-sis of RTC,tubular expression of caspase-3 and the content of MDA were increased in the UUO group compared with those in the sham-operated group,and were ameliorated significantly by PFD treatment(P<0.05).The activity of SOD was decreased in the UUO group,but was increased by PFD treatment(P<0.05).Our results showed that PFD could ameliorate TIF in the UUO group,and the possible mechanism was by reducing the apoptosis of RTC,which involved oxidative stress and caspase-3.
