Cyclin-dependent kinase inhibitor roscovitine attenuates liver inflammation and fibrosis by influencing initiating steps of liver injury.

Liver diseases present a significant public health burden worldwide. Although the mechanisms of liver diseases are complex, it is generally accepted that inflammation is commonly involved in the pathogenesis. Ongoing inflammatory responses exacerbate liver injury, or even result in fibrosis and cirrhosis. Here we report that roscovitine, a cyclin-dependent kinase inhibitor, exerts beneficial effects on acute and chronic liver inflammation as well as fibrosis. Animal models of lipopolysaccharide/D-galactosamine- and acute or chronic CCl4-induced liver injury showed that roscovitine administration markedly attenuated liver injury, inflammation and histological damage in lipopolysaccharide/D-galactosamine- and CCl4-induced acute liver injury models, which is consistent with the results in vitro. RNA-seq analysis showed that roscovitine treatment repressed the transcription of a broad set of pro-inflammatory genes involved in many aspects of inflammation, including cytokine production and immune cell proliferation and migration, and inhibited the TGF-β signaling pathway and the biological process of tissue remodeling. For further validation, the beneficial effect of roscovitine against inflammation was evaluated in chronic CCl4-challenged mice. The anti-inflammation effect of roscovitine was observed in this model, accompanied with reduced liver fibrosis. The anti-fibrotic mechanism involved inhibition of profibrotic genes and blocking of hepatic stellate cell activation. Our data show that roscovitine administration protects against liver diseases through inhibition of macrophage inflammatory actions and hepatic stellate cell activation at the onset of liver injury.

Liver diseases present a significant public health burden worldwide. Although the mechanisms of liver diseases are complex, it is generally accepted that inflammation is commonly involved in the pathogenesis. Ongoing inflammatory responses exacerbate liver injury, or even result in fibrosis and cirrhosis. Here we report that roscovitine, a cyclin-dependent kinase inhibitor, exerts beneficial effects on acute and chronic liver inflammation as well as fibrosis. Animal models of lipopolysaccharide/D-galactosamine-and acute or chronic CCl 4 -induced liver injury showed that roscovitine administration markedly attenuated liver injury, inflammation and histological damage in lipopolysaccharide/D-galactosamine-and CCl 4 -induced acute liver injury models, which is consistent with the results in vitro. RNA-seq analysis showed that roscovitine treatment repressed the transcription of a broad set of pro-inflammatory genes involved in many aspects of inflammation, including cytokine production and immune cell proliferation and migration, and inhibited the TGF-β signaling pathway and the biological process of tissue remodeling.
For further validation, the beneficial effect of roscovitine against inflammation was evaluated in chronic CCl 4 -challenged mice. The anti-inflammation effect of roscovitine was observed in this model, accompanied with reduced liver fibrosis. The anti-fibrotic mechanism involved inhibition of profibrotic genes and blocking of hepatic stellate cell activation. Our data show that roscovitine administration protects against liver diseases through inhibition of macrophage inflammatory actions and hepatic stellate cell activation at the onset of liver injury.
potency presented in the chronic liver injury animal model. The underlying mechanism involved NF-κB and MAPK pathway inhibition, repression of pro-inflammatory gene transcription and suppression of profibrotic genes and hepatic stellate cell activation. 3) These data suggest that roscovitine alleviates liver injury via inhibition of macrophage inflammatory actions and hepatic stellate cell activation, and provides evidence that evidence that therapeutic targeting of these driving factors by pharmacological inhibition of cyclin dependent kinases could be used as effective strategies for the treatment of inflammation-or fibrosis-involved hepatic diseases.

Introduction
Liver, as the central metabolic organ, is constantly exposed to pathogen-derived molecules, as a result of which it must remain in an immune tolerant condition to sustain homeostasis [1,2].
Injury to the liver, regardless of etiology, results in the disturbance of immune homeostasis and involves multiple cell types including hepatocyte, macrophages (Kupffer cells) and hepatic stellate cells (HSCs). Subsequent activation of signaling cascades initiates hepatic inflammation and tissue repair mechanisms. In pathological conditions, abnormal inflammatory responses can be fulminant or perpetual and result in various liver diseases including acute liver failure, steatohepatitis and liver fibrosis. Inflammatory cell infiltration is commonly involved in acute liver injury. Repetitive and/or sustained inflammation is an essential initiation factor leading to liver fibrosis, in which HSC activation and extracellular matrix (ECM) deposition cause hepatic tissue remodeling and fibrosis, or even promoting the development of cirrhosis and hepatocellular carcinoma (HCC) [3][4][5]. Therefore, the inhibition and treatment of uncontrolled liver inflammation is a crucial step in the treatment of liver diseases.
Cyclin-dependent kinases (CDKs) are serine/threonine protein kinases and are key regulators of the cell cycle and proliferation via regulation of phosphorylation state of various substrates involved in DNA replication and cell division [6]. Abnormal CDK activity and regulation are Alzheimer's disease [7][8][9][10] . Over the past decade, the broad-range purine analog CDK inhibitor roscovitine, also known as seliciclib or CY202, has been shown to exert promising inhibitory effect on various human cancers and has entered phase I and II clinical trials against multiple indications [11]. Apart from its potency against cancer, roscovitine also has anti-inflammatory properties. Roscovitine was proven effective in several in vivo models of inflammation including acute pleurisy, lung inflammation, and arthritis through the promotion of inflammation resolution and inhibition of proinflammatory cytokines transcription in macrophages [12][13][14]. The anti-inflammatory actions of roscovitine are thought to be due to its influence on immune cells: roscovitine promotes neutrophils apoptosis, blocks leukocyte extravasation and can downregulate the expression of pro-survival protein myeloid cell leukemia-1 at the transcription level [15]. Morever, roscovitine has potential therapeutic effects in the treatment of cystic fibrosis, systemic sclerosis, and tubulointerstitial fibrosis [16][17][18]. However, the effect of roscovitine on liver inflammatory diseases and fibrosis remains to be elucidated.
In this study, we identified that the CDK inhibitor roscovitine alleviates hepatic inflammation and fibrosis by inhibiting macrophage proinflammatory cytokine production, downregulating the TGF-β pathway and genes involved in inflammatory signaling pathways and leukocyte proliferation and infiltration, and suppressing profibrotic genes and HSC activation. These results indicate that roscovitine exerts protective effects in the onset of liver injury via suppression of macrophage inflammatory activities and HSC activation, and provides evidence that pharmacological inhibition of CDKs could be used as anti-inflammatory or anti-fibrotic therapies and may benefit patients with related liver diseases. Primary hepatocytes and Kupffer cells were isolated from C57BL/6 mice by two-step in situ liver perfusion protocols as previously reported [23]. Primary hepatocytes were grown in Hepatocyte Medium (Sciencell, America). Kupffer cells were cultured in RPMI-1640 supplemented with 10% FBS and 1% antibiotics (Penicillin-Streptomycin).

RNA-Seq analysis
Flash frozen mouse liver tissues from the LPS/GalN-induced liver injury model and TGF-β stimulated LX2 cells were used for total RNA extraction. The isolated RNA was used to RNA Integrity Number (RIN) value was used to assess the quality of the isolated RNAs. Only RNA sequences with RIN≥7.0 were used for sequencing. The sequencing reads were mapped to mm10 by STAR 2.5 and gene counting was quantified using feature counting software [24,25]. The DEseq2 R package was used for differential gene expression analysis [26]. The p value was adjusted by the Benjamini and Hochberg methods, and a 5% FDR cutoff value and fold change greater than 2 were set as the thresholds of the significant genes in the LPS/GalN-induced liver injury in vivo model, while the fold change threshold was 1.5 in the LX2 cells study. Gene set overlays were determined using BioVenn web tool [27].
Differentially expressed genes were further analyzed by gene-annotation enrichment analysis using the DAVID 6.8 bioinformatics platform and ClusterProfiler R package [28]. Gene Set Enrichment Analysis (GSEA) and leading edge analysis were performed using GSEA 4.0.3 software [29]. STRING 11.0 was used for network analysis, and enrichment analysis for GO was mapped onto the network [ Total protein was isolated from frozen liver tissues and cultured cells. Proteins were separated by electrophoresis, blotted and incubated with primary antibodies and corresponding secondary antibodies (anti-rabbit/mouse HRP conjugated antibody; PeproTech, China) for 1h.
Finally, protein bands were visualized using a chemiluminescence detection kit (Super signal, Thermo Scientific). These experiments were repeated for 2 or 3 times. Primary antibodies used in this study are listed Supplement Table 2.

Statistical analysis
All results are expressed as mean ± SD using GraphPad Prism 8.0 statistical software (GraphPad Software, Inc., La Jolla, CA, USA). Analysis of differences between different groups was performed via a two-tailed unpaired t-test. P < 0.05 was considered statistically significant.

Roscovitine reduces expression of inflammatory mediators in a dose-dependent manner in vitro
Roscovitine has shown a great anti-inflammatory potency in vitro [13,31].  Figure 2B). The gross liver morphology also appeared to be improved by roscovitine treatment ( Figure 2C). Histologically, H&E staining of liver tissues after LPS/GalN exposure revealed a significant degree of liver injury, characterized by extensive tissue structure destruction, congestion, inflammatory cell infiltration, and cell necrosis. This injury was remarkably ameliorated by roscovitine ( Figure 2D). TUNEL staining and its quantification consistently revealed that the increased hepatic cell death induced by LPS/GalN was reversed by roscovitine intervention ( Figure 2E). Collectively, these data indicate that roscovitine treatment significantly mitigates the degree of liver injury.
We next evaluated whether roscovitine alleviates hepatic inflammation. The extent of inflammatory cell infiltration was examined by IHC staining in liver sections. Macrophage (marked by F4/80) and neutrophil (marked by LY-6G) infiltration was markedly inhibited in roscovitine-treated mice compared with vehicle-treated mice ( Figure 3A). Abundant studies have proven that the extent of liver injury is closely related to the cytokine response; inhibition of inflammatory cytokines production is associated with alleviation of liver injury [32][33][34][35]. ELISA detection of TNF-α, IL-6 and IL-1β pro-inflammatory mediators in serum ( Figure 3B) and liver tissues ( Figure 3C)  mRNA expression of Il-6, Il-1β, and Tnf-α were markedly attenuated via roscovitine treatment ( Figure 3D). These data are consistent with the previous study that showed that roscovitine promoted clearance of inflammatory cells and resolution of inflammation [12].
These results indicate that in mice with acute liver injury treatment with roscovitine regulates inflammatory cell infiltration and inflammatory cytokine expression in the liver, contributing to suppression of inflammatory responses in vivo. Interestingly, the TGF-beta signaling pathway and biological process of tissue remodeling also showed significant down-regulation after roscovitine treatment ( Figure 4D). Members of these two gene sets are involved in regulation of liver disease progression, from initial liver injury caused by inflammation to liver fibrosis and cirrhosis. Downregulation of TGF-beta and other representative genes regulating tissue remodeling was validated by RT-PCR in vitro and in vivo ( Figure 4F). Taken together, these results show that roscovitine inhibits the transcription of a broad set of pro-inflammatory genes involved in many aspects in inflammation, and represses the TGF-beta signaling pathway and biological process of tissue remodeling, further ameliorating inflammation and tissue damage. Moreover, inhibition of the expression of genes involved in immune cell migration and proliferation might promote resolution of inflammation and reduce liver injury caused by immune cell infiltration during the course of chronic liver injury and inflammation.

Roscovitine attenuates liver injury and inflammation in the CCl 4 -induced acute liver injury model
The limitation of the LPS/GalN-induced acute liver injury model is that it largely depends on the early production of detrimental inflammatory mediators, leading to massive hepatocyte apoptosis and lethal outcomes at the late stage [38]. Therefore, the anti-inflammatory and hepatoprotective effects of roscovitine were further confirmed in the CCl 4 -induced acute liver injury model. H&E staining revealed massive centrilobular hepatic necrosis and inflammation in liver sections from CCl 4 -induced acute liver injury model mice ( Figure 5A). Moreover, serum ALT and AST levels were significantly increased in these mice after CCl 4 injection ( Figure 5B). These pathological changes were altered by roscovitine, suggesting alleviated liver damage. IHC staining showed that roscovitine treatment reduced F4/80 + macrophage and LY-6G + neutrophil infiltration in the CCl 4 -induced acute liver injury model ( Figure 5C).
Similarly, RT-PCR results demonstrated the suppression of Il-6, Il-1β and Tnf-α inflammatory genes expression in roscovitine-treated mice ( Figure 5D). Together, these data suggest that roscovitine exerts protective effects on inflammation and injury of liver parenchyma, in addition to attenuating inflammation-dominant liver injury.

Roscovitine protects against chronic CCl 4 -induced liver inflammation in mice
Based on the promising hepatoprotective efficacy of roscovitine in acute liver injury models, we hypothesized that roscovitine could exert similar protective effects against inflammation in a chronic liver injury model. We tested this hypothesis using a chronic CCl 4 -challenged mice model. At the endpoint of the experiment, CCl 4 -challenged mice demonstrated disturbed lobular architecture with inflammation and hepatocyte ballooning evidenced by H&E staining ( Figure 6A) and elevated serum ALT levels ( Figure 6B). The liver injury was accompanied by increased mRNA levels of proinflammatory cytokines including Il-6, Il-1β and Tnf-α in the CCl 4 -treated group ( Figure 6C). These pathological changes were moderately attenuated by roscovitine. Together, these data indicate that roscovitine demonstrates consistent anti-inflammatory properties in chronic CCl 4 -induced liver injury.

Roscovitine protects against liver fibrosis in vivo and in vitro
Repeated CCl 4 treatment induced the development of significant liver fibrosis, which was alleviated by roscovitine ( Figure 6D). Perpetual hepatic inflammation and inflammatory cells infiltration serve as crucial driving factors of HSC activation and liver fibrosis [39]. Moreover, activated HSC transdifferentiates to fibrogenic myofibroblasts characterized by enhanced ECM production, which is a crucial step in the development of liver fibrosis. Therefore, the anti-fibrotic effect of roscovitine was investigated by assessing its effect on inflammation and HSC activation. The expression levels of α-SMA and Col1a1 were assessed as dominant components of ECM and well-established markers of fibrosis and HSC activation [40]. IHC and western blot assays revealed significantly increased α-SMA and COL1a1 expression in the model group which was reversed by roscovitine treatment (Figure 6E, 6F, and S2D).
RT-PCR assessment of the expression levels of typical profibrotic markers was consistent with the IHC staining and western blot results ( Figure 6G). dose-dependent manner, indicating that roscovitine inhibits HSC activation ( Figure S3A). To profile gene expression change during HSC activation and roscovitine treatment, transcriptome analysis was performed in LX2 cells treated with TGF-β with or without roscovitine. A specific subset of profibrotic genes induced by TGF-β was suppressed by roscovitine ( Figure S3B). Further gene enrichment analysis revealed that genes effected by roscovitine are involved in many well-known biological processes that play critical roles in HSC activation and fibrosis including cell-matrix adhesion, focal adhesion assembly and the tumor necrosis factor-mediated signaling pathway ( Figure S3D). Notably, the expression of important marker genes in these gene sets was inhibited by roscovitine ( Figure S3C), suggesting that roscovitine is an effective inhibitor of HSC activation.
Taken together, our results show that liver inflammation and HSC activation were effectively inhibited by roscovitine.

Discussion
Liver disease can be caused by various etiologies, including alcohol, viral hepatitis and chemical insults and is an enormous global health burden [41,42]. Inflammation is a physiological response to injury and leads to subsequent secretion of inflammatory mediators involved in cellular defense and tissue repair [43]. Chronic injury left unchecked leads to persistence of liver inflammation and might result in tissue damage and disease progression.
Ongoing chronic inflammation induces the activation of macrophages to release proinflammatory and profibrotic mediators. TGF-β, predominantly produced by macrophages, activates HSCs and is generally considered the most potent fibrogenic cytokine [44].
However, there is a lack of efficient anti-inflammatory medications for liver diseases. The identification of effective pharmacotherapies for related hepatic diseases is urgently needed.
Roscovitine is a 2, 6, 9-substituted purine analogue that function as a potent inhibitor of CDK-1, -2, -5, -7 and -9 [6]. Recent studies have reported the protective role of roscovitine in lipoteichoic acid-induced lung inflammation and LPS-induced murine testis inflammation [45,46]. Although roscovitine has shown effective anti-inflammatory capacity in multiple by macrophage activation. Our data are consistent with those of previous studies that show that roscovitine promotes inflammation resolution by driving inflammatory cell apoptosis [12,47,48].
Inflammatory responses are thought to be regulated by multiple signaling pathways, and NF-κB and MAPK pathways play a predominant role in the innate immune response. NF-κB and MAPK contact with upstream Toll-like receptor 4 (TLR4), an LPS sensor, and become activated to initiate proinflammatory reactions [49,50]. Activated NF-κB directs the production of proinflammatory cytokines, particularly TNF-α, which is also by itself an activator of NF-κB, resulting in a positive feedback loop. Roscovitine administration down-regulates p-p65, p-IκB, and TNF-α expression levels, suggesting blockade of the NF-κB signaling pathway and its consequential activation. The MAPK pathway, which can also be activated by TNF-α, plays an essential role in stress and inflammatory responses [51].
JNKs and ERKs, two extensively-studied MAPKs, have a close relationship with macrophage-triggered inflammatory responses [52]. Deletion of JNK1 and JNK2 causes decreased polarization to the M1 phenotype in macrophages, indicating a pro-inflammatory role for JNK signaling [53]. The ERK1 and ERK2 is closely related to cytokine production regulation in macrophages [52]. As a CDK inhibitor, the cell death-inducing actions of roscovitine, such as antitumor properties and neutrophil apoptosis-inducing abilities, have been well studied. The role of roscovitine in the influence on the onset of liver injury has been studied to a lesser extent. Our results indicate that roscovitine can effectively suppress macrophage inflammatory actions and subsequent HSC activation, which are two preliminary steps of significant importance in liver injury initiation and progression. These data provide evidence that therapeutic targeting of these driving factors can be effective strategies for the treatment of inflammation-or fibrosis-involved hepatic diseases. However, additional research is required to translate these findings into a therapeutic tool that can prevent progression and deterioration in liver diseases.
Data availability statement: All supporting data are available by contacting the corresponding author.

Declaration of interests:
The authors have declared that no conflict of interest exists.      Livers of mice exposed to CCl 4 for 8 weeks were excised and processed for histological and biochemical investigations.