1. Selectively depletion of Loxl1 in HSCs attenuated the CDAA induced LOXL1 up-regulation in the whole liver
Based on our previously findings supporting LOXL1 expression in HSCs was strongly associated with liver fibrosis progression(9). To assess the crucial role of LOXL1 in HSCs during NAFLD development, we generated HSCs-specific Loxl1 deletion (Loxl1fl/flGfapcre) by crossing Loxl1fl/fl mice to mice bearing a Gfap-Cre transgene. Loxl1fl/fl mice were used as control in the following experiments.
Successful deletion of Loxl1 in Loxl1fl/flGfapcre mice was confirmed by qPCR analysis using isolated primary HSCs from the livers, accompanied by decreased Col1α1 and α-SMA expression (Fig. 1A). Six to Eight-week-old male Loxl1fl/flGfapcre mice and their littermate control mice (Loxl1fl/fl) were then subjected to CSAA or CDAA diet for 16 weeks (Fig. 1B). As expected, CDAA-fed the control mice led to significant increased LOXL1expression both in mRNA and protein levels in the whole liver compared with CSAA-fed control mice. However, this increase was abrogated in CDAA-fed Loxl1fl/flGfapcre mice (Figure C&D). In order to confirm deletion of LOXL1 in activated HSCs, we performed co-localization immunofluorescence of LOXL1 with α-SMA in CDAA-fed Loxl1fl/flGfapcre mice and Loxl1fl/fl mice. We found that LOXL1 expression in activated HSCs was significantly diminished in Loxl1fl/flGfapcre mice; interestingly, we found that LOXL1 also positioned between the plates of hepatocytes (Fig. 1E).
Altogether, these data suggested that increased LOXL1 was associated with CDAA-induced NAFLD progression, and we generated HSCs-specific Loxl1 deficiency mice with highly efficient disruption of LOXL1 in HSCs.
2. Selectively depletion of Loxl1 in HSCs decreased steatosis, ballooning and liver injury
To confirm the function of Loxl1 in HSCs during NAFLD development, we first investigated hepatic histopathological features in Loxl1fl/flGfapcre and Loxl1fl/fl mice challenged with CSAA or CDAA diet. In accord with previous observations(14), we found that CSAA-fed mice displayed nonpathological liver histology regardless of the genotype, whereas CDAA-fed mice developed NASH with remarkable hepatic steatosis, inflammation, and ballooning (Fig. 2A). In addition, we found that Loxl1fl/flGfapcre mice developed histologic features of NASH with a significantly lower nonalcoholic fatty liver disease activity score (NAS) with respect to control mice, including less steatosis (2.60 ± 0.16 vs. 1.60 ± 0.22, P < 0.05), less inflammation (2.40 ± 0.16 vs. 1.60 ± 0.22, P < 0.05) and less hepatocyte ballooning (2.10 ± 0.23 vs. 1.40 ± 0.16, P < 0.05) (Fig. 2.B). The liver weight/body weight ratio or spleen/body weight ratio was significantly increased in CDAA-fed control mice compared with CSAA-fed control mice, and this increase was slight decreased in CDAA-fed Loxl1fl/flGfapcre mice (P > 0.05) (Fig. 2C).
Consistent with histological observations, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were elevated in the CDAA-fed as compared to CSAA-fed control mice, however Loxl1 deficiency in HSCs markedly attenuated CDAA induced-elevation of ALT and AST activities by ~ 40–55% (p < 0.05, Fig. 2C&D), indicating a protective role of Loxl1 deficient against CDAA-induced NASH.
3. Selectively depletion of Loxl1 in HSCs reduced fibrosis in CDAA-induced NASH
To examine the NAFLD progression, we analyzed hepatic fibrosis using histological staining. Sirius red staining showed that CDAA feeding for 16 weeks induced typical pericellular fibrosis. Quantification of hepatic collagen deposition, indicated by SR and Collagen I staining, was markedly increased in CDAA-fed the control mice compared with CSAA-fed the control mice, whereas this rise was abrogated in CDAA-fed Loxl1fl/fl Gfapcre mice (Fig. 3. A-C). Consistent with histological evaluation, measurement of hydroxylproline level confirmed that liver fibrosis was less in CDAA-fed Loxl1fl/fl Gfapcre mice than in control mice (Fig. 3D). Furthermore, this resolved fibrosis in CDAA-fed Loxl1fl/fl Gfapcre mice was associated with decreased expression of fibrosis-related genes, such as α-SMA, Col1α1, Lox, Fbln5, Timp1, Mmp2, Mmp9, Mmp12, Mmp13, Tgfb1, Pai1, and Pdgfrb, as assessed by qPCR analysis, compared with CDAA-fed the control mice (Fig. 3E). Together, these findings suggested that CDAA-fed HSCs-specific Loxl1 deficiency mice exhibited less collagen deposition with decreased pro-fibrosis genes expression compared with CDAA-fed control mice.
4. Selectively depletion of Loxl1 in HSCs ameliorated liver inflammation and macrophage infiltration in CDAA-induced NASH
Next, we aimed to determine whether HSCs- specific Loxl1 deletion affected hepatic inflammation. We found that macrophage infiltration, indicated by CD68 staining, was much higher in the CDAA-fed control mice than that in the CSAA-fed the control mice, however this rise was abrogated in Loxl1fl/flGfapcre mice (Fig. 4A-B). Concordantly, this finding was supported by the decrease in hepatic mRNA expressions of the pro-inflammatory cytokines, such as Mcp1 and Tnfa expression in CDAA-fed Loxl1fl/flGfapcre mice compared with CDAA-fed control mice. A tendency towards a reduced expression was also observed for Il6, whereas no change was detected in Il10 mRNA (Fig. 4C). Collectively, these findings suggested that CDAA-fed HSCs-specific Loxl1 deficiency mice exhibited less macrophage infiltration with decreased pro-inflammation genes expression compared with CDAA-fed control mice
5. Selectively depletion of Loxl1 in HSCs reversed the metabolic abnormalities in CDAA-induced NASH
The above results suggested that deletion Loxl1 could reduce liver injury, inflammation and fibrosis in NASH. Furthermore, we aimed to detect whether Loxl1 deficiency in HSCs altered the metabolic phenotype or the steatosis in CDAA-fed mice.
We assessed the metabolic status of each group of mice. The initial body weight of all mice was comparable. Body weight during the whole study period was similar between CSAA-fed control mice and CSAA-fed Loxl1fl/flGfapcre mice, but was lower in CDAA-fed control mice in comparison with CSAA-fed mice. Notably, CDAA-fed Loxl1fl/flGfapcre mice reversed body weight loss to normal level (Fig. 5A). In line with body weight, epididymal fat to body weight ratio was higher approximately 20% in CDAA-fed Loxl1fl/flGfapcre compared with control mice (Fig. 5B).
Histologically hepatic steatosis, observed by Oil-Red O staining, was greater in CDAA-fed mice than CSAA-fed mice; in contrast, relatively low-level lipid deposition occurred in Loxl1fl/flGfapcre mice of CDAA feeding (Fig. 5C&D). This was also confirmed by biochemical analysis of hepatic triglycerides and NEFA (Fig. 5E), in contrast serum TG was increased in Loxl1fl/flGfapcre mice of CDAA feeding (Fig. 5F).
To evaluate the mechanisms by which selective Loxl1 deletion could influence the steatotic process, we detected several key lipogenic genes (ChREBP/Mlxipl, Srebp1c, Fasn, Scd1, Acc, Ppara). Consistent with a previous study, we also found that CDAA diet significantly down-regulated the expression of genes involved in fatty acid synthesis. These findings may be explained by compensatory hepatic uptake of serum lipids or by impairment in VLDL secretion from the live (16). Interestingly, a tendency towards a reduced expression was observed for these lipogenic markers between CSAA-fed Loxl1fl/flGfapcre mice and control mice, whereas no change was detected between CDAA-fed Loxl1fl/flGfapcre mice and control mice (Fig. 5G). Collectively, these findings suggested that selective deletion of Loxl1from HSCs reversed the metabolic disorder without effect of hepatic fatty acid synthesis.
6. Loxl1 deficiency in liver affected adipose tissue function in CDAA-induced NASH
Progressive adipose tissue dysfunction is key events in NASH development, and adipose transplantation could reverse the metabolic abnormalities associated with lipoatrophy(17), supporting the existence of an adipose tissue-liver crosstalk. Considerate Loxl1 deficient increase body weight and epididymal fat weight, we hypothesize that Loxl1 may affect the function of adipose tissue. To evaluate selective deletion of Loxl1 in liver crosstalk with adipose tissue, we performed the main adipikine (leptin, IL6, adiponectin) and the key lipogenic genes (Srebp1c, Mlxipl) in adipose tissue.
As expected, CDAA-fed the control mice caused an increase in Col1a1 in comparison with the control mice fed with CSAA diet. However, this indicator of ECM was lower in CDAA-fed Loxl1fl/flGfapcre mice than CDAA-fed the control mice (Fig. 6A).
Consistent with Srebp1c expression in liver, adipose tissues expression of Srebp-1C was significantly increased in CDAA-fed Loxl1fl/flGfapcre compared with CDAA-fed control mice, however no differences Mlxipl expression was observed (P > 0.05) (Fig. 6C). Interestingly, gene expression adipokines including leptin and IL6 was increased in CDAA-fed Loxl1fl/flGfapcre mice compared with CDAA-fed the control mice, a tendency towards an increased expression was also observed for adiponectin (Fig. 6.B). Serum leptin levels confirm this funding. These data suggested that HSCs-specific Loxl1 depletion reversed the metabolic disorder by recovery adipose tissue function characterized by increased fat mass, enhanced adipikine (leptin, IL6) releasing, and adipose tissue remodeling (Fig. 6.D).
7. LOXL1 levels were negatively associated with leptin levels in non-obesity NASH patients.
To assess the clinical relevance of our findings, we examined LOXL1 and leptin levels in NAFLD patients with no or mild or significant fibrosis stage (Table S2). Consistent with the mice data, we observed that serum LOXL1 level was significant higher in patients with no or mild fibrosis than those with significant fibrosis in NAFLD patients (F0/1 vs. F ≥ 2, 30.0 (21.9, 38.0) vs. 66.6 (31.0, 79.7) pg/ml, P < 0.05), a tendency of lower serum leptin levels in patients with no or mild fibrosis than those with significant fibrosis (F0/1 vs. F ≥ 2: 197.5(130.0, 251.0) vs. 132(90.0, 218.0) ng/ml, P > 0.05 ). A small sample size which may lead to results that potentially failed to be significant. Correlation analyses showed significantly reverse association between serum LOXL1 and Leptin in NAFLD patients (Fig. 7C). Furthermore, we noted that the prevalence of histologically detected with significant fibrosis was significantly higher among patients with non-obese NALFD (BMI < 25 kg/m2) (7/9, 77.8%) compared to those obese NAFLD (BMI ≥ 25 kg/m2) (4/14; 8.6%; p = 0.027). Together, our results demonstrated LOXL1 was associated with fibrosis during NAFLD development, meanwhile there will be a potential connection between LOXL1 and leptin in NAFLD, especially in non-obese NAFLD (Fig. 7D).