Mining host candidate regulators of schistosomiasis-induced liver fibrosis in response to artesunate therapy through transcriptomics approach

Background Artesunate (ART) has been reported to have an antifibrotic effect in various organs. The underlying mechanism has not been systematically elucidated. We aimed to clarify the effect of ART on liver fibrosis induced by Schistosoma japonicum (S. japonicum) in an experimentally infected rodent model and the potential underlying mechanisms. Methods The effect of ART on hepatic stellate cells (HSCs) was assessed using CCK-8 and Annexin V-FITC/PI staining assays. The experimental model of liver fibrosis was established in the Mongolian gerbil model infected with S. japonicum cercariae and then treated with 20 mg/kg or 40 mg/kg ART. The hydroxyproline (Hyp) content, malondialdehyde (MDA) content, superoxide dismutase (SOD) and glutathione peroxidase (GPX) activities in liver tissue were measured and histopathological changes of liver tissues were observed. Whole-transcriptome RNA sequencing (RNA-seq) of the liver tissues was performed. Differentially expressed genes (DEGs) were identified using bioinformatic analysis and verified by quantitative PCR (qPCR) and western blot assay. Results ART significantly inhibited the proliferation and induce the apoptosis of HSCs in a dose-dependent manner. In vivo, Hyp content decreased significantly in the ART-H group compared to the model (MOD) group and GPX activity was significantly higher in the ART-H group than in the MOD group. Besides, ART treatment significantly reduced collagen production (p <0.05). A total of 158 DEGs and 44 differentially expressed miRNAs related to ART-induced anti-schistosomiasis liver fibrosis were identified. The qPCR and western blot results of selected DEGs were consistent with the sequencing results. These DEGs were implicated in key pathways such as immune and inflammatory response, integrin-mediated signaling and toll-like receptor signaling pathways. Conclusion ART is effective against liver fibrosis using Mongolian gerbil model induced by S. japonicum infection. We identified host candidate regulators of schistosomiasis-induced liver fibrosis in response to ART through transcriptomics approach.


Introduction
Liver fibrosis is a very complex dynamic pathological process including hepatic stellate cells (HSCs) activation, immunity and inflammation, increased extracellular matrix (ECM) proteins and fibrogenesis, in which involved the activation or inhibition of multiple signaling pathways in various cell types [1].The major risk factors for liver fibrosis include hepatitis virus infection, S. japonicum infection, alcoholic liver disease, drugs and metabolic disorders [2].Numerous researches on the mechanism of liver fibrosis have focused mainly on the HSCs activation and thus activated HSCs has been considered to be antifibrotic target [3].Many drugs or small molecule are in clinical trials but less have been approved for the treatment of liver fibrosis in clinical [4].

Dozens of natural compounds demonstrate antifibrotic activities in various liver
fibrosis model in vivo and HSCs in vitro.Fuzheng Huayu (FZHY) can attenuate liver fibrosis by inhibiting HSCs activation and inflammatory response, regulation of ECM deposition, inhibition of hepatic sinusoidal capillarity and angiogenesis, in consequence, it has been approved by China food and drug administration for the treatment of liver fibrosis [5,6].Recently, Luo et al showed FZHY and ART could alleviate schistosomiasis liver fibrosis by modulating mitochondrial autophagy [7].
ART, a stable derivative of artemisinin extracted from the stems and leaves of artemisia annua, has many pharmacological effects including anti-inflammatory, anti-tumor and anti-fibrosis activities [8][9][10].Additionally, ART can inhibit fibrosis through different mechanisms involving inflammatory infiltration, autophagy of ferritin, collagen synthesis and HSCs apoptosis etc. [11][12][13].Several studies also proved that ART prevent schistosomiasis liver fibrosis by regulating mitochondrial function [7,14].At present, existing studies about anti-fibrosis mechanism of ART remain fragmented and unsystematic.
Mongolian gerbils are relatively suitable hosts for Schistosoma japonicum.The schistosomiasis hepatic fibrosis Mongolian gerbil model has stable fibrosis formation for the eggs continued slow stimulation, which is similar to the process of pathological injury of human liver [15].Additionally, unlike the New Zealand rabbit model, the Mongolian gerbil model does not experience slow self-healing in the stage of advanced liver fibrosis [16].It is the most realistic pathological reproduction of patients with infectious liver fibrosis.Compared with the liver fibrosis mice model, individual dynamics of biochemical indicators in Mongolian gerbil can be more easily tracked and individual differences between the models could be maximally homogenized.At the same time, it greatly facilitates consistent analysis of liver fibrosis pathological data.
We evaluated the effect of ART against liver fibrosis in HSCs (LX-2 cells) in vitro and schistosomiasis Mongolian gerbil model in vivo.Subsequently, we screened differentially expressed genes (DEGs) related to ART treatment by whole transcriptomics sequencing (RNA-Seq) of liver tissues to explore the potential regulatory mechanisms of ART against schistosomiasis liver fibrosis.

Ethical Approval of the Study Protocol
Animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Zhejiang Laboratory Animal Center (Zhejiang, China).

HSCs Proliferation and Apoptosis Detection
The antiproliferative effect of ART on HSCs was measured by CCK-8 assay.
Briefly, HSCs (2000 cells per well) were seeded in 96-well plate and exposed to five different concentrations of ART (10,20,40,60,80, 100 and 120 μM), and the control and blank group were treated with equal volume PBS.After incubation for 24 h, each well was supplemented with 10 μL CCK-8 solution and incubated for 1 h.
Cell viability was determined by detecting the absorbance at 450nm.Each experiment was performed in triplicate.
Apoptosis in HSCs was assessed by FITC/PI staining.Briefly, HSCs (1x10 4 cells per well) were seeded in 24-well plate and exposed to different concentrations of ART (40 and 80 μM) for 48 h.Cells were then added with the mixture of 195 μL Annexin V-FITC binding solution and 5 μL Annexin V-FITC, followed by addition of 10 μL PI pigment.After incubation for 10-20 min in the dark at 20-25℃, the cells were observed in the fluorescence microscope.

Animal Model and Artesunate Treatment
Forty clean grade male Mongolian gerbils (± 80g) were used in the experiment.
Cercariae were induced and collected by exposing infected water-immersed snails with Schistosomiasis to light for 1.

Whole Transcriptomics Sequencing
Total RNA was isolated form liver tissue by Trizol regent (Invitrogen, CA, USA).
Then RNA concentration and purity were measured by using Nano drop 2000.RNA integrity was identified by agarose gel electrophoresis and Bioanalyzer 2100 (Agilent, CA, USA) and considered to be good with RIN >7.0.Small RNA library was constructed according to protocol of TruSeq Small RNA Sample Prep Kits (Illumina, San Diego, USA) and single-end sequencing was performed on the Illumina Hi-seq 2500.For strand-specific library construction and paired-end sequencing on the Illumina Hi-seq 4000, RNA samples were purified according to the manuscript of the Epicentre Ribo-Zero Gold Kit (Illumina, San Diego, USA) and fragmented into small pieces.The cleaved RNA fragments were then reverse-transcribed to create the final cDNA library in accordance with the protocol for the RNA-Seq sample preparation kit (Illumina, San Diego, USA).

Data Processing and Bioinformatics Analysis
Raw reads were processed by Cutadapt to genenrate clean reads and verified through FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/).Clean reads were then mapped to the genome of mice and assembled using StringTie.
Transcriptomes from Mongolian gerbil samples were merged to reconstruct a comprehensive transcriptome using perl scripts.The transcript-level expression level of RNA-seq was analyzed by calculating FPKM.DEGs were selected with log2 (fold change) >1 or log2 (fold change) < -1 and with statistical significance (p value < 0.05) by R package Ballgown.GO terms and KEGG pathways of these DEGs were annotated.Protein-protein interaction (PPI) network of DEGs was performed by the STRING online analysis tool (https://string-db.org/),PPI network was visualized and analyzed by Cytoscape software 3.7.2,and the TOP30 DEGs were selected as Hub genes.For miRNAs analysis, unique sequence with length in 18~26 nucleotide was mapped to specific species precursors in miRBase 21.0 by BLAST search to identify known miRNAs and novel 3p-and 5p-derived miRNAs.Target genes were predicted by TargetScan 8.0 and Miranda 3.3a.

Real-time Quantitative PCR
To validate RNA-Seq results, total RNA was extracted from liver tissue using Trizol regent.Reverse transcription for each sample was performed on 200 ng of total RNA by using cDNA reverse transcription kit (Takara, Beijing, China).
Thereafter, real-time fluorescent quantitative PCR was utilized to detect the expression levels of the candidate genes.The primers were designed with Primer 5.0 software (S1 Table ) and synthesized by Tsingke Biotechnology Co., Ltd.Results were calculated by the 2 -ΔΔ Ct method and GAPDH were used as an internal reference gene for each example.

Statistics Analysis
Data were presented as the mean ± SEM.Statistical analyses were performed using SPSS 26.0 and Graph Pad Prism 8.4 software.Statistical comparison between groups was performed using one-way analysis of variance (ANOVA) with a Tukey test and student's t test.P<0.05 was considered statistically significant.

Effect of Artesunate on HSCs and Animal Model
The effect of ART on HSCs was investigated by CCK-8 and FITC/PI double staining assay.Compared with control group, apoptotic morphological changes were observed after ART treatment under the microscope.Results showed that ART (10 μM) significantly inhibited HSCs viability in proliferation at 24 h after treatment (p<0.05 vs. control) (Fig 1A and B).With the concentration of ART increased, an increasing number of apoptosis cells were observed (Fig 1C). Figure 2A illustrates the respective timeline of the in vivo studies.In vivo, serum HA and ALT level were detected respectively after 8 weeks of infection and 12 weeks of ART treatment.In the ART-H group, HA and ALT serum level decreased after ART treatment although the differences were not statistically significant.Additionally, the GPX activity were significantly higher in the ART-H group than that of in the MOD group (Fig 2B).In appearance, the livers in the MOD group were chestnut black and had slightly increased volume, blunt edges.There was a marked improvement in liver and spleen morphology and color in the ART-H group whereas changes to liver and spleen index were not significant (data were not shown).Histopathological changes in livers were accessed by H&E and Masson staining.Compared with CON group, inflammatory cell infiltration and collagen fiber deposition were observed in the MOD group, while collagen deposition decreased significantly in the ART-H group, indicating that high-dose of ART administration have protective effect against liver fibrosis (Fig 2C).

Fibrosis in Response to Artesunate
For each sequencing library, the reads of Q20, Q30 and GC content exceeded 99%, 96% and 47% respectively, which indicated a high quality of clean data (S2 Table ).
At the same time, more than 90% of the total reads were successfully mapped to the reference genome, of which more than 64% of readings are uniquely located (S3 Table ).Then uniquely mapped reads were used for subsequent analysis.DEGs were then screened for subsequent analysis.Compared with CON group, 467 genes were up-regulated and 144 genes were down-regulated in the MOD group, 187 genes were up-regulated and 142 genes were down-regulated in the ART group.Compared ART group with MOD group，117 genes were up-regulated and 216 genes were

Discussion
ART has achieved more attention because of its various pharmacological properties.Studies have shown that ART has significant anti-fibrosis activity, while the specific molecular mechanism has not been systematically elucidated [17].We investigated the effect of ART on liver fibrosis in HSCs and infectious Mongolian gerbil model.Mechanistically, RNA-Seq analysis of liver tissue was performed to explore the molecular mechanism of ART on liver fibrosis.
ART can significantly inhibit the proliferation and induce apoptosis of HSCs in vitro and alleviate effectively schistosomiasis liver fibrosis in vivo in this study.In HSCs, ART significantly inhibited cell proliferation in a dose-dependent manner within a concentration range from 10μM to 120μM.In addition, the number of apoptotic cells gradually increased with the increased concentration of ART, indicating that ART promotes their apoptosis in a dose-dependent manner.The latest research also showed that ART promotes HSCs apoptosis by inhibiting mitochondrial function [14].In order to explore the anti-fibrosis activity of ART in vivo, we successfully constructed the liver fibrosis experimental model in Mongolian gerbil infected with S. japonicum.The liver index were improved slightly by ATR gavage treatment, and pathological changes of liver tissue showed that inflammation cells infiltration and collage deposition were reduced, and similar findings have been reported from one previous study suggesting a decreased area of fibrosis and granuloma in ART intervention group in the mouse model [14], which indicating that ART relieves schistosomiasis liver fibrosis to some extent.
To further explore the anti-fibrosis mechanism of ART, we performed transcriptome RNA-Seq of liver from CON, MOD and ART group.Preliminary cluster analysis revealed slight individual gene expression differences in MOD group, which may be caused by the fact that the number of infected schistosome cercariae was not strictly controlled.Further bioinformatics analysis determined 158 DEGs related to ART anti-schistosomiasis liver fibrosis.GO function analysis showed that these DEGs were involved in immune response, extracellular space and calcium ion transportation terms.KEGG pathway analysis revealed that immune response, focal adhesion, platelet activation and HIF-1 signaling pathway were significantly enriched.
Current strategies of treatment of liver fibrosis are directly mainly to inhibit HSC activation, induce activated HSC apoptosis, increase ECM degradation, and suppress inflammation (Fig 5).Activation of HSCs is considered to be a key event in liver fibrosis and TGF-β1 is responsible for HSCs activation.Src family tyrosine kinases are activated in different organs fibrosis such as lung, kidney and liver, and considered to be the amplifiers of TGF-β1 signal in the pathological process of fibrosis [18].The activated Src under hypoxic conditions of tissue cells can mediate the expression of Ctgf induced by TGF-β1, further stimulate collagen secretion and eventually accelerate fibrosis process [19,20].At present, Src has become a potential therapeutic target for various organ fibrosis.For example, Sarakatinib, a tyrosine kinase activity inhibitor, has been approved by the FDA for the treatment of idiopathic pulmonary fibrosis [21,22].Our RNA-seq results showed that Src is significantly down regulated in ART group compared to MOD group, which indicted that Src is a potential target of ART to liver fibrosis [23] (Berkoz et al. 2021).
Additionally, previous research reported that ART prevent schistosomiasis liver fibrosis by regulating mitochondrial function in HSC [7,14].We identified 6 mitochondrial-related differentially expressed genes (Ddit4, Ndufa7, Higd2a, Ucp2, Mrpl36, Fahd1) in this study, which are potential targets of ART.The DNA damage-induced transcript Ddit4 is expressed under stress and is located downstream of HIF-1α, which negatively regulates mTOR-induced cell proliferation and induces autophagy [24,25].Ctss is one of the TOP30 DEGs in this study and it promotes cell migration and invasion by regulating Ca 2+ homeostasis and ECM degradation [26].Studies have shown that inhibiting Ctss could increase the expression of mitochondrial calcium uniporter and enhance the absorption capacity of mitochondrial Ca 2+ , leading to mitochondrial Ca 2+ overload, the production of large amounts of ROS, and finally cell apoptosis mediated by mitochondria [27].
However, further experimental verification in vivo is required to confirm this interesting hypothesis.
Excessive deposition of ECM is the main pathological feature of liver fibrosis, and thus increasing ECM degradation is a pivotal strategy for antifibrotic therapies.
Therefore, Ctgf may be a potential intervention target of liver fibrosis reverse.

Conclusion
We developed liver fibrosis model in Mongolian gerbil of mimicking human infection with S. japonicum cercariae.The efficacy test showed that ART is effective in preventing liver fibrosis in vitro and in vivo.ART may alleviate effectively schistosomiasis liver fibrosis by decrease inflammation and collagen fibers deposition.We established the first comprehensive transcription profile, mined host candidate regulators and provided an overview of the complex network of the mechanism of Schistosomiasis liver fibrosis in response to ART.It is essential for understanding the effective mechanism of ART and for verifying their potential as diagnostic markers or novel therapeutic agents for Schistosomiasis liver fibrosis.
5 h and counted by direct observation under a light microscope.To established the animal model of schistosomiasis, 30 Mongolian gerbils were each infected with 80 S. japonicum cercariae by the abdominal patch method.Eight weeks post-infection, Mongolian gerbils infected with cercariae were administered by oral gavage with praziquantel diluted in 1% CMC-Na solution at the dose of 75 mg/kg• d -1 for 3 days.Twelve weeks post-infections, 30 Mongolian gerbils infected were randomly divided into 3 groups, model infection group (1% CMC-Na, MOD), low-dose ART treatment group (20mg/kg• d -1 , ART-L) and high-dose ART treatment group (40mg/kg• d -1 , ART-H).Mongolian gerbils in ART-L and ART-H group were treated by oral gavage with 0.2 mL ART solution with corresponding concentration; Mongolian gerbils in normal control (CON) and MOD group were administrated by oral gavage with 0.2 mL 1% CMC-Na solution, once daily for 12 weeks.Histological Assessment and Biochemical Indicators Detection Inflammation and fibrosis of liver were accessed by histological analysis and biochemical indicators of HA, ALT, malondialdehyde (MDA) and glutathione peroxidase (GPX) detection.Superior segment of left lateral lobe of each Mongolian gerbil liver was fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin-eosin (H&E) and Masson's trichrome for light microscopy.The degree of inflammation and fibrosis was graded according to the Blueprint for the Diagnosis and Assessment of Therapeutic Efficacy in Liver Fibrosis Due to Schistosomiasis, and Consensus on Evaluation of the Diagnosis and Efficacy of Hepatic Fibrosis in 2019.Serum HA/ALT levels were detected by using automatic biochemical analyzer to evaluate liver injury and liver function.Levels of MDA and activity of GPX in liver tissue were detected by spectrophotometer.

Fig 1 .
Fig 1.The effect of Artesunate on HSCs.(A) HSCs morphology after 24 h of ART intervention with different concentrations under an inverted phase-contrast microscope (100X); (B) Viability of HSCs incubated with different concentrations of ART for 24 h; (C) Fluorescence microscopy images of apoptosis cell after 24 h of ART treatment with different concentration of ART (a: 40 μM and b: 80 μM), 100X.Data are presented as mean ± SEM (n=3); ***p<0.001(ART treatment vs control

Fig 2 .
Fig 2. The effect of Artesunate on Schistosoma-induced liver fibrosis in Mongolian gerbil.(A) Experimental timeline of in vivo; (B) Levels of HA, ALT, GPX and MDA; (C) Liver appearance and pathological changes of liver tissue (200X), HE staining and Masson staining score statistics.Data were presented as mean ± SEM (n=4 per group).*p<0.05(after administration vs before administration).

Fig 4 .
Fig 4. GO and KEGG enrichment and PPI analysis of differentially expressed genes.(A) GO enrichment analysis; (B) KEGG enrichment analysis; (C) (left) protein-protein interaction analysis, blue: down-regulation, red: up-regulation; (right) Hub gene screening, interactions degree_top30 as Hub genes, dark red represents a high degree of connectivity.

Fig 5 .
Fig 5. Potential candidate regulators of Schistosomiasis liver fibrosis in response to Artesunate therapy.The red arrows indicate upregulated genes and the green arrows indicate downregulated genes; asterisk indicates genes reported previously.