Preventive effect of isostrictiniin from Nymphaea candida on carbon tetrachloride-induced hepatic fibrosis in mice

Abstract This study aimed to investigate the preventive effect of isostrictiniin (ISO) from Nymphaea candida against CCl4-induced hepatic fibrosis (HF) in mice. HF in mice was induced by intraperitoneally injecting 10% CCl4 olive oil solution twice a week. After intragastric administration for 8 weeks. ISO (25, 50, or 100 mg/kg) could significantly reduce the serum AST, ALT, ALP, HA, LN, PC-III, C-IV, IL-1β, and TNF-α levels in HF mice, and the collagen fiber deposition in ISO group was significantly less than that in the model group. Compared with control group, the protein expression of IKKα, NF-ҡB p65, p-NF-ҡKB p65, p-IҡBα in model group were increased as well as the relative content of IKKα, NF-ҡB p65, and IҡBα mRNA. ISO could reverse the expression of these relative proteins and mRNA in HF mice. Our findings confirm that ISO has anti-HF activity, and its mechanism is related to the regulation of NF-ҡB pathway. Graphical Abstract


Introduction
Hepatic fibrosis (HF) is a resulting pathological process of the development of various liver diseases such as steatohepatitis, hepatitis B, and autoimmune hepatitis (Campana and Iredale 2017). Persistent fibrosis can gradually evolve into liver cirrhosis and liver cancer if left treatment. More than one million people worldwide died of liver fibrosis leads to endstage liver disease each year(Wand 2018). Therefore, it is very important to prevent hepatic fibrosis in the course of chronic liver disease to cirrhosis. Hepatic fibrosis is a reversible pathological process and is a compensatory reaction in the process of liver injury and tissue repair after inflammation stimulated by various pathogenic factors (Maurizio and Massimo 2019). In recent years, more and more studies have shown that hepatic fibrosis and even early cirrhosis can be reversed if treated in time. Traditional Chinese medicine (TCM) shows better efficacy in the process of anti-liver fibrosis, and active components derived from TCM (resveratrol, tanshinone II, etc) have also demonstrated remarkable antihepatic fibrosis activity with little untoward effects (Yu 2019).
Nymphaea candida is a herbaceous hydrophyte growing in lakes and swamps in Xinjiang of China, and its flowers has been used as a folk medicine for treatment of hepatitis (Liu 1999). Hepatoprotective activity of N. candida has been verified by our previous experiments (Zhao 2011;Zhang 2016). Isostrictiniin (ISO, Figure S1) is a polyphenol compound isolated from this plant, and possess antioxidant and hepatoprotective activities (Maierdan et al. 2018 ;Zhao 2021). However, whether ISO can attenuate hepatic fibrosis is unclear. Based on these, this study aimed to investigate anti-hepatic fibrosis effects of ISO and its mechanisms by CCl 4 -induced hepatic fibrosis in mice for the development and application of this compound.

Results and discussion
Hepatic fibrosis induced by CCl 4 is widely used in the preparation of animal fibrosis models (Yanguas 2016). Single-dose administration of CCl 4 can lead to steatosis and necrosis of liver cells, and long-term administration can lead to fibrosis, cirrhosis, and liver cancer. CCl 4 is metabolised by cytochrome P450-dependent mixed oxidase in hepaticendoplasmic reticulum to active trichloromethyl radicals (CCl 3 Á) and chlorine radicals (ClÁ), leading to lipid peroxidation and membrane damage (Weber 2003;Marques 2012). In this study, subcutaneous injection of CCl 4 was used to induce liver fibrosis in mice. After 8 weeks of administration, HE staining and Masson staining found that the mice liver was damaged and fibrotic, indicating that the liver fibrosis model was successfully prepared. Furtherly, the anti-hepatic fibrosis activity of ISO was studied by CCl 4 -induced hepatic fibrosis in mice.
The body weight gain of mice in the positive control colchicine (COL, 0.1 mg/kg) and ISO (25, 50, 100 mg/kg) groups were significantly increased compared to model group ( Figure S2A). In the model group, the liver was dark red, dull and yellowish in color, tough texture, and accompanied by ascites and nodules, which adhered to the surrounding lobes and blurred edges, and ISO (25, 50, 100 mg/kg) could improve this appearance liver to different degrees ( Figure S2B). The liver index in the ISO groups (25, 50 and 100 mg/kg) was remarkably lowered compared with model group (P < 0.01) ( Figure S2C). However, ISO and COL have not showed significant reduction on elevatory spleen index in mice (P < 0.01) ( Figure S2C). These results suggested that ISO treatment could alleviate hepatic injury in mice.
As the indicators of evaluating liver function, serum ALT, AST and ALP levels can reflect the degree of liver injury. When liver cells are damaged, intracellular ALT, AST, and ALP are released into the blood and elevating the levels of ALT, AST, and ALP in the serum (Jamjute 2009). In this study, Serum AST, ALT and ALP activities as well as liver HYP level were changed significantly in mice receiving CCl 4 twice a week for 8 weeks and terminated 16 h later (P < 0.01). Conversely, the serum AST, ALT and ALP levels were decreased by ISO (25, 50 and 100 mg/kg) (P < 0.01) compared with those in the model group. These results showed that ISO could ameliorate liver function that was damaged by CCl 4 (Figure S3).
At present, PC-III, Col-I, HA and LN are regarded as biomarkers for hepatic fibrogenesis in clinic (Zhan 2016). In addition, HYP also can be used to represent the degree of fibrosis . The metabolic collagen HA, LN, PC III and C-IV levels were determined in liver tissue of HF mice ( Figure S4). These collagenic markers were aberrantly increased in liver tissue of CCl 4 induced fibrotic mice, and their levels is higher than those in control liver of mice (P < 0.01). Following ISO treatment, the elevated contents of HA, LN, PC III and C-IV in liver tissue could be attenuated in varying degrees compared with model group. (P < 0.05 or P < 0.01). Compared to the model group, the HYP content in liver tissues in mice could be decreased by ISO but no statistics significance (P > 0.05). These data reveal that ISO can alleviate ECM accumulation caused by the chronic live injury and postpone the development of liver fibrosis.
Hepatic fibrosis was evaluated histologically using HE and Masson staining in the different groups. As shown in Figure S5A or S5B, liver tissue slices in the control group showed exhibited the normal lobule architecture, central veins located within radiating hepatic plate, and accompanied by abundant hepatocytes. Compared with control group, liver tissue in the model group were showed severe pathological changes such as disordered lobular structure, steatosis, obvious inflammatory cell infiltration, hydropic degeneration, spotty necrosis and inflammatory infiltration. Treatment with ISO (25, 50 and 100 mg/kg) appeared to reverse those pathological changes to differing extents. To investigate whether ISO can attenuate ECM accumulation in CCl 4 -induced mice, Masson staining was used to evaluate collagen accumulation in liver. The result was shown in Figure S5C or S5D, in the model group, a large number of false lobules formed by blue fiber bands encircled the portal area, and cuoles formed by necrotic liver cells were observed. ISO could significantly reduce the collagen secretion and liver fibrosis level in liver tissues, and the blue fiber bands around the portal area were significantly reduced compared with the model group. Therefore, ISO presented remarkable effect in ameliorating hepatic fibrosis, in maintaining the intact structure of hepatic lobuli and reducing the formation of collagen deposition showed reduced degree of hepatic inflammation and fibrosis.
The activation of HSCs induces changes in their morphology, the loss of their retinoid droplets and increases in the a-SMA expression. Hence, a-SMA is considered as a sensitive and specific indicator for the activation of HSC. As shown in Figure S6, the expression of a-SMA was significantly inhibited by ISO (25, 50, 100 mg/kg) administration in a dose-dependent manner (P < 0.01). These results shows that ISO has the antiinflammatory and improving fibrosis activities in the damaged liver.
As a kind of transcription factor, NF-jB exists in in a variety of hepatocytes such endothelial cells, kupfferer cells and parenchymal cells, and plays an important role is in the hepatic fibrosis signaling pathway (Luedde and Schwabe 2011). IjB stably binds to NF-jB through its C-terminus specific ankyrin repeat motif at rest. NF-jB is prevented from transferring into the nucleus, and presents in the cytoplasm without activity. However, when hepatocytes are stimulated by proinflammatory factors, IjB is phosphorylated by the IjB kinase complex (IKK) and leads to its degradation. Free NF-jBp65 was released and rapidly translocated into the nucleus to regulate the inflammatory response of target genes (Zheng 2019). Pro-inflammatory cytokine including IL-6 and TNF-a were activated, and formed an inflammatory microenvironment within the liver, and further promote the progression of liver fibrosis (Seki and Schwabe 2015 ;Algandaby 2016). The NF-rB signal pathway was activated by CCl 4 , and the expression of IKKa, NF-jBp65, p-NF-jBp65 and p-IjBa proteins were upregulated in the mice liver tissues, while the expression of IjBa protein was downregulated. As shown in Figure S7, with GAPDH as an internal reference, compared with control group, the expression of IKKa, NF-ҡB p65 and activated p-NF-ҡB p65, p-IҡBa protein in liver tissue of the model group mice increased(P < 0.05), while the expression of Iҡ Ba decreased(P < 0.05), and ISO (25, 50, 100 mg/kg) groups could significantly ameliorate the expression of NF-jB related proteins in liver tissue. The results showed that ISO dose-dependently prevented the nuclear translocation and expression of IKKa and NF-kB p65, while also inhibiting both their phosphorylation and degradation of IjBa. All of these results suggest that ISO exerts an inhibitory effect in relation to the activation of the NF-jB signaling pathway in fibrotic liver tissues. Moreover, ISO (25, 50, 100 mg/ kg) could significantly reduce the release of TNF-a and IL-6 compared with model group (P < 0.01) ( Figure S7). The results indicated that ISO can inhibit the release of pro-inflammatory cytokines by regulation of NF-jB signaling pathway to achieve antiliver fibrosis effect.

Conclusions
ISO can significantly attenuate CCl 4 -induced liver fibrosis, and its effective mechanism may be attributed to reduction of ECM accumulation, decrement of collagen synthesis and regulation of NF-jB signaling pathway. These results indicate that ISO may offer a reference into the development of drugs for the prevention and treatment of liver fibrosis.

Disclosure statement
The authors have declared no conflicts of interest.

Funding
This work is supported by grants from National Natural Science Foundation of China (NSFC) (81760631).