武大-基础医学院-李红良-Nature Medicine(2018)

概要：作者利用多组学方法，发现ALOX12在肝脏缺血损伤中上调，进而产生12-HETE（代谢组测序），12-HETE结合GPR31（已经报道为12-HETE的受体，但功能研究不多）引发肝脏炎症反应，加重肝损伤。

创新：1. ALOX12-12HETE-GPR31轴在肝脏缺血再灌注损伤中作用首次发现；2. 多组学应用；3. 除啮齿动物（小鼠），作者还在其他大型动物模型，包括猪、恒河猴中验证了该轴的作用；3. GPR31作为G蛋白偶联受体，有着良好的药物转化价值（extensive pathological relevance and distribution in almost all cell types; possess molecular pockets for small-molecule binding)。

主要研究内容：

1. Upregulation of ALOX12 in the ischemic stage

Figure 1 ALOX12 is dramatically upregulated during hepatic IR injury. (a) flow chart. (b) cluster analysis. (c) KEGG pathway enrichment analysis. (d) Circos plot showing the correlations between the proteomics and DGE data. Histograms with a blue border at the outer edge of the circle represent correlations with a −log10P value, and only correlations with P < 0.05 (by correlation analysis with Pearson correlation coefficient) extend to the light blue area. Correlation tracks are shown inside the circular layout as follows: red bars represent positive correlation (r > 0), and bars located in the light red area indicate r values larger than 0.50; green bars represent negative correlations, and the light green area indicates an r value lower than −0.50 by correlation analysis with Pearson correlation coefficient. (e) Heat maps of mRNA expression of members in the AA metabolism pathway. (f) Relative Alox12 mRNA expression levelin the livers of mice. (g) The relative mRNA expression of ALOX12 in the livers of individuals. (h) ALOX12 protein expression levels. (i) ALOX12 protein expression in the livers of individuals. B, baseline; I, ischemia; R, reperfusion. (j) (k) immunohistological staining of ALOX12.  For statistical analysis, a two-tailed Student’s t-test (f) or one-way ANOVA with Tamhane’s T2 (M) post hoc analysis (g) was used.

构建缺血再灌注模型，在缺血前、缺血1h、再灌注3h、6h、12h、24h收集小鼠肝脏用于转录组和蛋白组测序，发现在众多生物学过程中，脂质代谢变化最明显，并筛选出花生四烯酸代谢通路中变化最显著的ALOX5基因。

2. 12-HETE is the primary metabolite affected by changes in ALOX12 expression during ischemia–reperfusion 

Figure 2 ALOX12 directly promotes 12-HETE accumulation in hepatic IR injury. (a) Heat map of major AA metabolites catalyzed by lipoxygenases or cytochrome P450 enzymes in the livers of mice. n = 10 mice in the sham group, n = 15 mice in other groups. (b) Time-dependent changes in the profiles of relative ALOX12 protein expression, 12-HETE content and AA content in the livers of mice (n = 6 mice in each time point). (c) Relative protein expression level of ALOX12 and 12-HETE content in the cell lysates of primary hepatocytes after 10, 30 and 60 min of hypoxia. n = 3 independent experiments. (d,e) ALOX12 protein expression in cell lysates of primary hepatocytes following Alox12 knockdown (d) or Alox12 overexpression (e) by adenovirus under the condition of normoxia or hypoxia for 1 h. (f,g) 12-HETE content in the cell lysates of primary hepatocytes treated as in d (f) and e (g). (h) 12-HETE content in the livers (left) and serum (right) of Alox12-KO and WT controls at 6 h after reperfusion or in the sham group. (i) 12-HETE content in the livers (left) and serum (right) of Alox12-HTG and NTG control mice in the sham and 6-h reperfusion groups. (j) 12-HETE content in the livers (left; n = 17) and serum (right; n = 19) of human individuals subjected to hepatic IR injury. For statistical analysis in f–j, one-way ANOVA with Tamhane’s T2 (M) post hoc analysis was used. 

从众多花生四烯酸代谢产物（由LOX和CYP450催化）中，作者筛选出变化最明显的12-HETE（羟基廿碳四烯酸)。12-HETE不仅在缺血前后变化明显，还与ALOX12在肝缺血再灌注中的含量变化呈相似趋势；在肝脏中敲减或过表达ALOX12，12-HETE的含量也随之改变。进一步的模式动物实验证明（ALOX12-KO和ALOX12-HTG（肝脏特异性表达）），12-HETE是含量受ALOX12表达量影响。最后的人群实验也证实了，12-HETE在缺血前后含量发生变化。

那么ALOX12表达量变化是否影响了IRI表型呢？

3. Manipulation of Alox12 expression affects the degree of ischemia–reperfusion-induced liver injury

Figure 3 Alox12 knockout inhibits IR-induced liver dysfunction, cell death and inflammation. (a) ALT and AST levels in the serum of mice.n = 10 mice in each group. (b,c) H&E (b) and TUNEL (c) staining of liver sections from WT mice and Alox12-KO mice. n = 4 mice per group at each time point with 24 images for each mouse. NA, necrotic area. (d) Representative western blot showing protein expression of BCL2, BAX and cleaved caspase-3 (c-CASP3) in the livers of mice. n = 6 mice for each group. (e) Tumor necrosis factor (TNF) and CCL2 content in the serum of mice. n = 4 mice in sham WT group and n = 6 mice in other groups. (f) The relative mRNA expression levels of cytokines and chemokines in the livers of mice. n =4 mice per group. (g) Representative images of immunofluorescent staining of CD11b+ (left) and Ly6G+ (right) inflammatory cells (red) in liver sections from mice. n = 4 mice in each group with 24 images for each mouse. Scale bar, 20 µm. (h) Activation of NF-κB signaling in the livers of mice. n = 6 mice for each group; n = 3 western blots for each band. For statistical analysis, one-way ANOVA with Bonferroni’s post hoc analysis (for TNF in e and Tnf in f) or with Tamhane’s T2 (M) post hoc analysis (for a, CCL2 in e and Il6 and Cxcl2 in f) was used.

动物表型验证：ALOX12 KO鼠构建缺血（60min）再灌注（6h）模型，检测了肝功能（a,血浆ALT AST）、HE染色（梗死面积）、TUNEL染色（凋亡）、WB凋亡蛋白（Bcl2抗凋亡，BAX促凋亡，C-caspase3)、血清和肝脏炎症因子变化、炎症细胞浸润和NF-kB通路 。再ALOX12-HTG小鼠中做了同样的验证（相反的表型）——supplementary FIG3.

那ALOX12的下游产物12-HETE的变化是否影响肝脏IRI表型？

4. 12-HETE promotes hepatic ischemia–reperfusion injury via induction of a burst of inflammation

Figure 4 12-HETE promotes hepatic IR injury by inducing a burst of inflammation. (a) Left, Pearson correlation coefficient between Alox12 or 12-HETE and the coexpression modules (represented by the colored boxes at the left of the table) determined based on WGCNA using the DGE data in Figure 1a (n = 6 groups with 5 mice in each). Right, KEGG analysis results for genes in the yellow coexpression module that were significantly enriched in the indicated inflammatory pathways (P < 0.05 by Fisher’s exact test). (b) Correlations between the expression of Alox12 or the level of 12-HETE and the expression of core genes related to the pathways listed in a. The correlation was visualized in a user-defined manner with line width and color saturation; stronger correlations are indicated by thicker lines and darker colors. (c,d) Representative western blots (n = 3 western blots for each band) showing phosphorylated and total p65, JNK, p38 and ERK protein expression (c) and qPCR results showing relative mRNA expression levels for Il6, Tnf and Ccl2 (d) in primary hepatocytes treated with 12-HETE for the indicated periods. n = 3 independent experiments with 2 replicates each. (e–g) 12-HETE content (e), phosphorylated and total levels of p65, JNK, p38 and ERK (f) and relative mRNA expression of cytokines and chemokines (g) in primary hepatocytes treated with ML355 (10 µM) under normoxia (Nor) or hypoxia (Hyp) conditions. For western blot analysis.n = 4 independent experiments for e; n = 3 independent experiments with two replicates each for f and g. For statistical analysis,one-way ANOVA with Bonferroni’s post hoc analysis (for Ccl2 in d and for e and g) or with Tamhane’s T2 (M) post hoc analysis (for Tnf and Il6 in d) was used.

WGCNA: 加权基因共表达网络分析——用来描述不同样品之间基因关联模式的系统生物学方法，可以用来鉴定高度协同变化的基因集, 并根据基因集的内连性和基因集与表型之间的关联鉴定候补生物标记基因或治疗靶点。该分析方法旨在寻找协同表达的基因模块(module)，并探索基因网络与关注的表型之间的关联关系，以及网络中的核心基因。一般可应用的研究方向有：不同器官或组织类型发育调控、同一组织不同发育调控、非生物胁迫不同时间点应答、病原菌侵染后不同时间点应答。

用WGCNA分析方法，作者找出了与ALOX12表达和5HETE水平呈最高程度相似性的一组基因集，并且他们再多种炎症信号通路上有明显富集。无论缺氧细胞模型还是IRI动物模型，高表达ALOX12促炎（NA-kB和MAPK信号激活，炎症因子表达增加），敲减ALOX12抗炎（supplymentary FIG4)。且单独给与12-HETE干预原代肝细胞也表现出促炎表型，而抑制12-HETE生成（ML355 10um——ALOX12抑制剂）减轻炎症。

下一步：机制探索

5. GPR31 is responsible for 12-HETE-mediated hepatic ischemia– reperfusion injury

Figure 5 GPR31 is responsible for 12-HETE-mediated hepatic IR injury. (a) The major pathways contributing to 12-HETE function determined by GSEA of RNA-seq results from primary hepatocytes treated with 12-HETE (100 nM) under normoxia or hypoxia conditions. The red area in each numbered circle indicates the number of overlapped leading-edge genes (the core genes that account for their gene set’s enrichment signal) induced by 12-HETE under normoxia and hypoxia. Analysis included data from four independent experiments. (b) Schematic of GO analysis showing that GPCR signaling is a major contributor to 12-HETE function. The data used in this GO analysis were from the leading genes highlighted in the red areas of the circles in the RNA-seq data shown in a and Supplementary Figure 5a with the heaviest 5% of edge weights (n = 4 samples from independent experiments for each group). The 15 most significantly enriched pathways (P < 0.05 by Fisher’s exact test; edge number > 100) are shown. (c) Levels of phosphorylated and total PKC and JNK in GPR31-, GPR40-, GPR75- or GPR120-knockdown cells treated with 12-HETE or vehicle control for 1 h. n = 3 independent experiments. (d) Dot blot representing pairwise GSEA comparison of RNA-seq data among 12-HETE-treated hepatocytes (n = 3 in each group). Dot color indicates that the gene signature set is enriched in the group with corresponding font color. NES, normalized enrichment score. (e) Left, GSEA showing the merged gene sets of genes in the pathways shown in Figure 5d. Right, Venn diagram showing shared leading genes between two comparisons. Data were statistically analyzed by GSEA based on three independent experiments. (f) ALT and AST content in the serum of mice. (g) Representative images of TUNEL staining (green) of liver sections at 6 h after reperfusion of mice. n = 4 mice for each group with 24 images for each mouse. Scale bar, 20 µm. (h) Relative mRNA expression of inflammatory cytokines and chemokines (n = 4 mice in each group) in the livers of mice. For statistical analysis, one-way ANOVA with Tamhane’s T2 (M) post hoc analysis (f) or a two-tailed Student’s t-test (h) was used.

为了进一步明确12-HETE产生促炎作用的机制，作者又对12-HETE处理的原代肝细胞进行了转录组测序分析（四组：nor_veh, nor_12hete, hyp_veh, hyp_12hete），在nor_12hete和hyp_12hete组找出了共同显著变化的基因，GSEA分析表明这些基因参与炎症相关过程。关于b图的解释没有看得很明白，不确定原文的意思是否是参照Fig4a的方式寻找overlap gene，然后从这些基因中最重的5%权重边（？）被用于基因本体(GO)分析，富集到了GPCR通路。

确定验证哪些GPCR的方法

接着用腺病毒敲减备选的4个GPCR观察12HETE干预情况下PKC-JNK激活情况，发现只有GPR31 knock down抑制了12-HETE介导的PKC-JNK激活，以此证明GPR31可能是12-HETE的下游。作者又构建了GPR31KO和GPR31-res肝细胞模型，用12-HETE干预后进行转录组测序分析，做进一步验证（d，e这里的分析方法没读得明白）。作者还建立了GPR31 KO的基因鼠，并进行了肝脏IR造模，测了上述一样的表型，都缓解了。

由于12-HETE对于炎症的表型上述都是在细胞层面，而阻断12-HETE是否可以缓解IR损伤还需要在动物水平做进一步验证。

6. Blocking 12-HETE production inhibits hepatic ischemia– reperfusion injury in mice and pigs

这里应用了两种ALOX12抑制剂（ML355和CDC）去抑制12-HETE产生， 发现ML355的抑制效应更明显，且其缓解肝脏IRI的效应比CDC更好，呈剂量依赖性的减轻IRI，在整个IRI周期都发挥作用。

由于小鼠模型并不能很好的反应人体情况，作者又用了大型动物（pig）做同样的效应验证，表明ML355减少5-HETE表达，缓解IRI损伤。

7. ML355 inhibits liver damage in a nonhuman primate model of hepatic ischemia–reperfusion

Figure 6 ML355 inhibits liver damage in a monkey hepatic IR model. (a,b) Serum ALT, AST and ALP levels of human subjects after liver resection (a, n = 38 individuals) and of monkeys subjected to 60-min ischemia followed by periods of reperfusion of varying duration (b, n = 5 in each group). w, weeks. compared to the corresponding baselines by one-way ANOVA with Tamhane’s T2 (M) (a) and with Bonferroni’s post hoc analysis (b). (c) Serum ALT and AST levels (n = 5 in vehicle group, n = 6 in ML355 group) in monkeys treatedwith ML355 (3 mg per kg body weight) or with vehicle after 60 min of hepatic ischemia followed by the indicated periods of reperfusion. *P < 0.05, **P < 0.01, ***P < 0.001 compared to corresponding time points of vehicle group by two-tailed Student’s t-test. (d) Representative images of H&E staining on monkey liver sections in ML355- and vehicle-treated groups after 48 h of reperfusion. n = 5 monkeys per group with 24 images for each monkey. Scale bar, 200 µm for H&E staining (10×); 20 µm for H&E staining (40×). (e) Relative mRNA expression (normalized to that of 18S rRNA) of inflammatory mediators in the IR-challenged hepatic lobes and control lobes of monkeys in ML355- and vehicle-treated groups at 48 h after reperfusion. n = 4 monkeys in the vehicle-treated group and n = 5 monkeys in the ML355-treated group. *P < 0.05, **P < 0.01, ***P < 0.001 by one-way ANOVA with Bonferroni’s post hoc analysis. (f) Representative perfusion CT images of livers from monkeys treated with vehicle or ML355 at 48 h, 1 week and 2 weeks after reperfusion. n = 5 monkeys at each time point for each group. Each image was generated from 39 scans in each monkey. HAP, hepatic arterial perfusion; HPP, hepatic portal perfusion. (g) Representative images of H&E staining of heart sections from mice treated with ML355 or vehicle control in the sham group or at 7 d after MI surgery. n = 4 mice for each group with 2 consecutive sections for each mouse. Scale bar, 1 mm. (h) Representative images of 2,3,5-triphenyl-2H-tetrazolium chloride (TTC) staining of serial brain sections of mice at 24 h after stroke. n = 8 mice in each group with 7 consecutive sections for each mouse.

为了更好的临床转化，作者用了更接近人类的恒河猴模型对ML355对IRI的效应做了进一步的验证。