FABP4抑制剂BMS309403通过调控FABP4/PPAR-γ信号轴减轻肝肺综合征大鼠肝肺损伤OA

Objective Hepatopulmonary syndrome(HPS)is characterized by inflammation,pulmonary vasodiation and angiogenesis.Elevated serum level of fatty acid binding protein 4(FABP4)in patients with liver cirrhosis is associated with poor prognosis,and FABP4 is involved in the regulation of inflammation and angiogenesis.Using a bile duct ligation(BDL)-induced HPS rat model and the FABP4 inhibitor BMS309403 for intervention,this study aims to investigate the role of the FABP4/PPAR-γ signaling axis in the pathogenesis of HPS and to evaluate the protective mechanism of FABP4 inhibition against hepatopulmonary injury.Methods A total of 24 male SPF SD rats(6 to 8 weeks old,weighing 200 to 220 g)were enrolled and randomly divided into(n=6):a sham operation group(Sham group),the BDL postoperative 3-week group(BDL3W group),the BDL postoperative 5-week group(BDL5W group),and a BDL+BMS309403 treatment group(BDLB group).The BDL3W,BDL5W and BDLB groups underwent BDL to establish an HPS model,while the Sham group underwent only bile duct manipulation without ligation.The BDLB group received intraperitoneal injections of BMS309403(5 mg/kg,twice per week)from postoperative day 15 for 3 weeks.The BDL3W group was sacrificed at week 3 post-surgery,while the Sham,BDL5W,and BDLB groups were sacrificed at week 5 post-surgery.Histopathological changes in the liver and lung tissues were evaluated using HE staining.The severity of hepatic fibrosis was assessed by Masson's trichrome staining,while hepatic lipid accumulation was examined using Oil Red O staining.Microarray was performed on lung tissues from the Sham and BDL3W groups,and integrated analysis of differentially expressed genes(DEGs)was conducted,including:heatmap plotting based on specific gene sets,visualization of differential expression with volcano plots,and GO functional enrichment analysis.Immunohistochemistry was used to detect the localization and expression levels of CD31,iNOS,TNF-α,NF-κB p65,and PPAR-γ in lung tissues.Immunofluorescence staining was performed to determine the expression of FABP4.The protein levels of FABP4 and PPAR-γ in lung tissues were quantified by Western blotting.Arterial blood gas analysis was conducted to measure PaO2 and P(A-a)O2 for the evaluation of pulmonary function,and serum levels of ALT and AST were determined to evaluate hepatic function.Results ① Compared with the BDL3W group,the BDL5W group exhibited progressively disorganized liver structure,gradually aggravated fibrosis,and a significant increase in relative collagen areas(P<0.01).Serum ALT levels increased continuously from the Sham group to the BDL3W and BDL5W groups(P<0.01),with AST levels showing the same trend(P<0.01).In the lungs,inflammatory cell infiltration aggravated progressively,and PaO2 decreased gradually from the Sham to BDL3W and BDL5W groups(P<0.05).Compared with the BDL3W group,P(A-a)O2 was markedly elevated in the BDL5W group(P<0.01),confirming the successful establishment of the HPS model.② In the BDL3W group,FABP4 gene was significantly upregulated in the lung tissues(FC≥1.5,P≤0.05).GO enrichment analysis revealed significant enrichment of lipid metabolism-related genes,suggesting that FABP4 and lipid metabolic processes play important roles in the pulmonary pathogenesis during HPS.③Compared with the BDL5W group,the BDLB group showed significantly decreased serum AST and ALT levels(P<0.01),accompanied by attenuated hepatic fibrosis,as evidenced by a significant reduction in relative collagen area(P<0.01).Pulmonary inflammatory infiltration,hemorrhage,and atelectasis were alleviated,with a significant increase in PaO ₂(P<0.01)and an obvious decrease in P(A-a)O ₂(P<0.01).④ Immunofluorescence and Western blotting revealed that FABP4 expression in the lung tissues of the BDLB group was significantly lower than that in the BDL5W group(P<0.05).Immunohistochemistry and Western blotting further demonstrated that PPAR-γ expression in the lung tissues was significantly enhanced in the BDLB group compared with the BDL5W group(P<0.05).⑤ Compared with the BDL5W group,the BDLB group exhibited reduced hepatic lipid droplet accumulation.Immunohistochemical analysis showed a significant decrease in the diameter of pulmonary microvascular lumens(P<0.01),along with significantly downregulated expression of iNOS(P<0.01),and remarkably reduced expression levels of the inflammatory factors TNF-α and NF-κB p65(P<0.01).Conclusion The FABP4 inhibitor BMS309403 ameliorates hepatopulmonary pathological injury and improves hypoxemia in HPS rats by modulating the FABP4/PPAR-γ signaling axis,ameliorating hepatic lipid metabolism disorder,inhibiting NF-κB p65/TNF-α-mediated pulmonary inflammatory response,and suppressing iNOS/NO-driven pulmonary microvascular dilation.