Therapeutic potential of natural products in schistosomiasis-associated liver fibrosis

Schistosomiasis is a parasitic disease that endangers human health and social development. The granulomatous reaction of Schistosoma eggs in the liver is the main cause of hepatosplenomegaly and fibrotic lesions. Anti liver fibrosis therapy is crucial for patients with chronic schistosomiasis. Although Praziquantel is the only clinical drug used, it is limited in insecticide treatment and has a long-term large-scale use, which is forcing the search for cost-effective alternatives. Previous research has demonstrated that plant metabolites and extracts have effective therapeutic effects on liver fibrosis associated with schistosomiasis. This paper summarizes the mechanisms of action of metabolites and some plant extracts in alleviating schistosomiasis-associated liver fibrosis. The analysis was conducted using databases such as PubMed, Google Scholar, and China National Knowledge Infrastructure (CNKI) databases. Some plant metabolites and extracts ameliorate liver fibrosis by targeting multiple signaling pathways, including reducing inflammatory infiltration, oxidative stress, inhibiting alternate macrophage activation, suppressing hepatic stellate cell activation, and reducing worm egg load. Natural products improve liver fibrosis associated with schistosomiasis, but further research is needed to elucidate the effectiveness of natural products in treating liver fibrosis caused by schistosomiasis, as there is no reported data from clinical trials in the literature.


Introduction
Schistosomiasis is a parasitic disease, in which trematodes infections poses a serious threat to human health and social development.Schistosomiasis is found in 78 countries in the tropics and subtropics and is predominantly endemic in sub-Saharan Africa (WHO, 2022).Intestinal schistosomiasis and urogenital schistosomiasis are two primary pathologies caused by Schistosoma infections in humans, with the former being primarily caused by S. japonicum and S. mansoni and the latter by S. haematobium.(McManus et al., 2018).Worms parasitize the veins of human hosts to mate and lay eggs are excreted in feces or urine.These eggs hatch as miracidia and infect intermediate species-specific host snails in fresh water.After 4-6 weeks, the eggs develop into infectious cercariae that can penetrate human skin and cause disease.Acute infections occur mostly in locals, travelers and immigrants and present symptoms of transient urticaria rash, allergic pneumonia, and Katayama syndrome.(Clerinx and Van Gompel, 2011).The progression of these infections can be characterized by chronic abdominal pain, loss of appetite, and liver polyps, and eventually lead to hepatosplenomegaly, portal hypertension, ascites, gastrointestinal varices, and even life-threatening gastrointestinal bleeding (Colley et al., 2014).Currently, the only clinically effective drug for treating schistosomiasis is praziquantel (Kabuyaya et al., 2023).However, due to the incomplete efficacy of praziquantel and the potential for drug resistance, there is an urgent need to find cost-effective alternatives or complementary treatments (Santana et al., 2021).More research is crucial to investigate novel targeting mechanisms of schistosome-induced liver fibrosis in order to discover new drugs or praziquantel analogs that can treat schistosomiasis.

Pathogenesis of chronic schistosomiasis
Fibrosis of the liver and portal system is the main pathological manifestation of intestinal schistosomiasis and is the result of an immune response caused by the invasion of schistosome eggs into the liver and blood vessels.The deposition of eggs causes a granulomatous inflammatory response mediated by CD4 T lymphocytes + , which is characterized by a markedly active Th2 immune response, such as increased levels of cytokines and chemokines, and the recruitment of lymphocytes, neutrophils, eosinophils, macrophages, and fibroblasts, followed by extracellular matrix (ECM) and collagen fibril production of liver tissue, with the eggs being trapped in the liver and unable to be excreted, finally resulting in a fibrotic inflammatory infiltrate forming around the eggs (Chiu et al., 2003;Amaral et al., 2017;Ho et al., 2022).The expedited Th2 response during the schistosomiasis infection may be related to alternate activation of macrophages.During the chronic infection phase, alternatively activated macrophages (M2 macrophages) are stimulated by IL-13, IL-33, IL-4, and ROS to regulate the expression of Arg1, IL-10, and TGF-β1, which act directly or indirectly on hepatic stellate cells and contribute to α-SMA and collagen production, leading to liver fibrosis (Peng et al., 2017;Tan et al., 2018;Yu et al., 2021).The soluble egg antigen (SEA) can also activate M2 macrophages via STAT6 and PI3K signaling pathways or directly activate hepatic stellate cells via the P38/JNK MAPK signaling pathway (Liu P. et al., 2013;Tang et al., 2017).In addition, the deposition of eggs significantly reduced the enzymatic activities of O 2 .
-and H 2 O 2 detoxification, by superoxide dismutase (SOD), catalase, and glutathione peroxidase (GSHPx) and increased the levels of hepatic products from lipid peroxidation, which may stimulate the progression of liver fibrosis (Gharib et al., 1999).

Natural products against schistosomiasis-associated liver fibrosis
An increasing number of studies have demonstrated that bioactive ingredients of medicinal plants are a promising alternative to current clinical therapy.The current direction of anti-schistosomiasis drug research is focused on the screening of compounds with therapeutic targets and the development of praziquantel analogs.Anti-fibrotic treatment is essential for patients with chronic schistosomiasis as even deworming does not completely stop the progression of liver fibrosis (Bergquist et al., 2017;LoVerde et al., 2021).In schistosomiasis-endemic countries such as China, Brazil, Zimbabwe, and Kenya, the antischistosomiasis pharmacological effects of natural products or plant extracts have been extensively studied in an attempt to discover alternative drugs (Molgaard et al., 2001).Currently, the active mechanism of various natural products in the treatment of schistosomiasis-associated liver fibrosis has been reported, which may modulate fibrotic factors such as IL-13, growth stimulation expressed gene 2 (ST2), TGF-β1, TNF-α and anti-fibrotic factors such as IL-10, Tregs, MHC II through intracellular signaling pathways such as NF-κB pathway, PI3K/AKT pathway and TGF-β1/Smad pathway (Liu et al., 2014;Tang et al., 2017;Kamdem et al., 2018;Huang et al., 2020).

Natural compounds
Based on the diversity that biological exploration of natural products provides for drug discovery, the active substances of natural products and their independent pharmacological effects in mixtures have attracted much attention (Phillipson, 2001;Simoben et al., 2018).Traditional medicinal plants are highly diverse and many metabolites have been shown to have therapeutic effects on liver fibrosis in schistosomiasis (Table 1).

Artemisinin and its derivatives
Artemisinin is a sesquiterpene lactone derived from Artemisia annua L., and artemisinin-based combination therapies (ACTs) are widely used to treat malaria.Artemisinin including its derivatives such as artesunate, dihydroartemisinin, and artemether has also been shown to have pharmacological effects such as anti-cancer, antiviral, anti-inflammatory, and anti-parasitic (Ho et al., 2014).Artemisinin and its derivatives have been shown to kill helminths in animal models (rabbits, rats, dogs) with worm reduction rates ranging from 41% to 98%, which is based on its high lethality to larvae and females (You et al., 1992;Xiao et al., 1994;Gold et al., 2017;Correa et al., 2019).In Schistosoma mansoni infected mice, the combination of artesunate (400 mg/kg) and praziquantel (500 mg/kg) significantly decreased hepatic P53 expression and increased Bcl-2 expression (Hegazy et al., 2018).Lower doses of artemether (50 mg/kg), artemether reduced the number of eggs in mice's feces, whereas higher doses of 400 mg/kg artemether greatly reduced the number and diameter of liver granulomas (Lescano et al., 2004;El-Beshbishi et al., 2013).A possible mechanism for this reduction is the inhibition of the expression of schistosomal metabolic enzymes such as glycolytic key enzymes as well as thioredoxin glutathione reductase (TGR), cytochrome c peroxidase (CcP) and SOD (Zhai et al., 2000;Abdin et al., 2013;El-Lakkany and Seif El-Din, 2013).Furthermore artemether has been shown to mediate a shift from a Th2 to a Th1 response in schistosomes, characterized by an increase in IFN-γ levels and a decrease in IL-4 and IL-10 levels (Madbouly et al., 2015).Interestingly, Keiser et al. found that artemether did not rely on synergy with the immune response for its anti-schistosomal effects, even though immunomodulation was beneficial in suppressing egg-induced hepatotoxicity (Keiser et al., 2010).In summary, artemisinin and its derivatives may be a potential treatment for schistosomiasis liver fibrosis.

Chlorogenic acid
Chlorogenic acid, 5-O-caffeoylquinic acid (5-CQA), a natural polyphenolic compound, is widely found in various fruits, vegetables, and medicinal plants, such as apples, eggplants, coffee beans, Lonicera japonica Thunb.And Eucommia ulmoides Oliv.It is most abundant in green coffee beans.It has significant protective effects on cardiovascular, gastrointestinal, liver, nerve, and metabolism due to its antioxidant, antibacterial, and anticancer biological activities (Naveed et al., 2018;Lu et al., 2020).Chlorogenic acid has been reported to protect against different types of liver fibrosis through NOX/ROS/MAPK, ERK/Nrf2, TLR4, and NF-κB pathways (Shi et al., 2013;Shi et al., 2016;Yuan et al., 2017;Wei et al., 2018).Chlorogenic acid inhibited the elevated expression of TGF-β receptor I, CTGF, and α-SMA after IL-13 treatment of LX2 cells in a dose-dependent manner, ranging from 56 μM to 225 µM.In vivo studies in Schistosoma japonicum-infected mice have shown that the use of chlorogenic acid (5-20 mg/kg for 4 weeks) reduces IL-13 expression and significantly reduces the size of liver granulomas (Wang et al., 2017).Further mechanisms suggest that IL-13 affects miR-21/Smad7 signaling, which in turn affects liver fibrosis.IL-13 is a key factor in the progression of schistosomiasis disease which is consistent with the view of Wynn et al. (2004).Furthermore, 56-225 µM chlorogenic acid also directly interferes with miR-21-regulated TGF-β1/ Smad7 signaling, which reduces the expression of CTGF, TIMP and MMP-9 and decreases collagen and ECM deposition (Yang et al., 2017).

Curcumin
Curcumin (1,7-bis-(4-hydroxy-3-methoxyphenyl)-hepta-1,6diene-3,5-dione), is a natural polyphenolic compound, mainly extracted from the rhizome of Curcuma longa L. Curcumin is the key active component of turmeric, showing antioxidant, antiinflammatory, anti-cancer, anti-microbial, and tissue (heart, nerve, liver) protective effects (Sohn et al., 2021;El-Saadony et al., 2022).The protective effects of curcumin against different types of liver injury are mainly mediated by reducing lipid peroxidation, activating the Nrf2 signal and inhibiting NF-κB activity (Khan et al., 2019).Curcumin (50-200 mg/kg) was found to upregulate MMP-1 and inhibit TIMP-1, resulting in a reduction in liver granuloma volume by up to 79% and collagen content by 38.6% (Li et al., 2007).Low expression of GSH, GST, SOD, and CAT caused by S. mansoni infection increased significantly after 2 weeks of treatment with 40 mg/kg curcumin (Abu Almaaty et al., 2021).A total dose of 400 mg/kg curcumin treatment was found to suppress serum levels of IL-12 and TNF-α in the infected group, possibly related to immune regulation triggered by inhibition of NF-κB activity (Jagetia and Aggarwal, 2007;Allam, 2009).Additionally, it significantly raised the mRNA expression of PPAR while reducing TGF-β1 (Chen et al., 2009).These studies suggest that curcumin may be an effective in the treatment of schistosomiasis-induced liver fibrosis.

Paeoniflorin
Paeoniflorin is the main active ingredient of the Paeonia lactiflora Pall., a monoterpene glycoside compound used in the treatment of cancer, depression, diabetes, liver disease, and autoimmune disorders (Ma et al., 2020;Zhang and Wei, 2020).Mouse peritoneal macrophages that are stimulated to produce TGF-β1 by SEA, causing the promotion of the proliferation of HSC and the synthesis of collagen.Chu et al. demonstrated for the first time that 7.5-120 mg/L of paeoniflorin (colchicine, positive control, 1 µM) selectively downregulated the level of Smad3 phosphorylation through TGF-β1 signaling and inhibited the proliferation of HSC (Chu et al., 2007).Another study found that 30 mg/kg paeoniflorin significantly reduced schistosome-induced elevated levels of IL-13 and decreased STAT6 phosphorylation levels and collagen I expression by increasing SOCS-1 expression (Li et al., 2010).Further studies showed that 100 μg/mL paeoniflorin directly or indirectly inhibited alternative activation of Kupffer cells by reducing JAK2 and STAT6 phosphorylation (Chu et al., 2011).Paeoniflorin may be a promising drug for the treatment of fibrosis in schistosomiasis.

Plant extracts
The roots, stems, leaves, flowers, and fruits of plants are processed using certain technological methods to obtain herbal bioactive ingredients that affect diseases.

Green tea extract
Camellia sinensis (L.) Kuntze is a perennial woody plant whose young leaves and flowers are processed into beverages or medicines (Butt et al., 2015).Green tea has been widely demonstrated to have preventive effects against diabetes, cancer, and cardiovascular disease.Its health properties are attributed to the bioactive polyphenols contained in it, particularly catechins (Xing et al., 2019).Epigallocatechin gallate contains 30%-50% of green tea catechins and is known for its potent antioxidant, anti-obesity, anti-inflammatory, anti-cancer, and other pharmacological activities (Yang et al., 2020).Bin Dajem et al. showed that green tea at a concentration of 3% (w/v) reduced hepatocellular necrosis and perivascular collagen fibers by decreasing lipid peroxidation, but failed to significantly improve liver function (Bin Dajem et al., 2011).Another study found Matcha (a Japanese green tea powder made from finely powdered dried tea leaves) to have lower levels of polyphenols and higher levels of caffeine, quercetin, and rutin than traditional green tea (Ramez et al., 2021).Matcha (3 g/kg b. w) contained more theanine and rutin than other green teas, reducing TNF-α, IFN-γ, and IL-13 levels, increasing IL10 levels, which led to the inhibition of the development of liver granulomas, the restoration of SOD, CAT and GSH-Px activity as well as MDA and TAC levels through antioxidant capacity (Kochman et al., 2020).The natural components of green tea may be a promising complementary treatment therapy for schistosomiasis.

Boswellia serrata resin extract
Frankincense is the resin that exudes from the bark of the Boswellia Roxb.tree, a member of the olive family, and boswellic acid is the most important triterpenoid of frankincense, especially 3-O-acetyl-11-keto-β-boswellic acid (β-AKBA) (Al-Harrasi et al., 2021).Liu et al. combined frankincense oil resin extract with cyclodextrin (BSE-CD) to address hydrophilicity issues.They first found that liver egg granulomas formed by the eggs of S. japonicum contained high levels of leukotriene B 4 .BSE-CD (280 mg/kg for 3 weeks) significantly reduced the size of liver granulomas, possibly caused by the reduced expression of MMP-9, LTB 4 , and PGE 2 (Liu M. et al., 2013).Further postulated mechanisms suggested that it could reduce the inflammatory response around eggs by inhibiting NF-κB signaling and reducing the expression of VEGF, TNF-α, and MCP-1 in mice (Liu et al., 2014).

Ampelopsis grossedentata extract
Ampelopsis grossedentata (Hand.-Mazz.)W.T. Wang, also known as vine tea, is a plant of the genus Ampelopsis in the family Vitaceae, mainly distributed in southern China.The pharmacological effects of vine tea are mainly summarized as anti-inflammatory and analgesic, hepatoprotective, hypotensive, hypolipidemic, antitumor, and anti-aging (Xie et al., 2019;Tong et al., 2020;Wang et al., 2023).Flavonoids are the main efficacy components of vine tea, which includes dihydromyricetin, myricitrin, myricetin, quercetin, rutin, and kaempferol (Xu et al., 2012).Dihydromyricetin has the largest content with a mass fraction of 34% and is considered to be foundational for the health benefits of vine tea (Feng et al., 2018;Zhang Q. et al., 2021).The total flavonoids of vine tea have been proven to have anti-liver fibrosis effects (Li et al., 2022).Ampelopsis grossedentata extract containing 90% dihydromyricetin (150 mg/kg for 8 weeks) significantly ameliorated hepatic fibrosis in S. japonicuminfected mice, which was superior to praziquantel alone.(Fang et al., 2010).30 μM Dihydromyricetin significantly inhibited the activation of HSC-T6 cells in vitro, mediated through the promotion of AMPK phosphorylation and inhibition of the TGF-β1/Smad signaling pathway (Zhang et al., 2018).In vivo, it (100, 200, 400 mg/kg) downregulated TGF-β1/Smad signaling, improved liver function and reduced ECM deposition.Colchicine was used as a positive control at a dose of 0.2 mg/kg (Liang et al., 2019).In addition, another active ingredient, myricetin was shown to have a toxic effect on S. japonicum worms through induction of apoptosis, with an LC50 of 600 μM at 24 h.Interestingly, it (at 250 mg/kg) reduces the number of worms and eggs as well as the size of liver granulomas by modulating the immune response (lowering the ratio of Th2 and Th17 cells) (Huang et al., 2020).

Ginger extract
Ginger (Zingiber officinale Roscoe), a perennial herb of the ginger family, is a medicinal plant with the same origin as food, and has the effect of promoting sweating and relieving symptoms, causing a warming sensation of the body, and suppresses vomiting (Zhang M. et al., 2021).Ginger crude aqueous extract (500 mg/kg) slowed the development of granulomatous inflammatory infiltrates and reduced hepatic egg load after schistosome infection, which was more pronounced after treatment with ginger-derived nanoparticles (Mostafa et al., 2011;Abd El Wahab et al., 2021).This may be related to the powerful antioxidant effect of ginger extract and its ability to scavenge free radicals, as evidenced by the restoration of CAT activity and MDA levels.Another study showed that ethanolic extract of ginger also inhibited oxidative stress and inflammatory mediators to improve schistosomiasis-associated liver fibrosis (Aly and Mantawy, 2013).Interestingly, Sanderson et al. suggested that the ethyl acetate extract of ginger (150 mg/kg) did not kill the egg load and helminth load of schistosomeinfected mice, which was attributed to the alternative extraction solvent and the varying treatment doses of the extracts (Sanderson et al., 2002).Given the lack of data on the role of ginger extract as a treatment for schistosomiasis-associated liver fibrosis, further study is required.

Conclusion
The main objective of this paper is to summarize the mechanistic studies of selected metabolites and plant extracts for the treatment of schistosomiasis-associated liver fibrosis.Some metabolites or plant extracts that did not address pharmacological mechanisms or were partially uncommon were excluded from the review.In addition, in the literature reviewed reported inconsistent ranges in dosage and varying assessment criteria for fibrosis.
Praziquantel is currently the most commonly used medication for schistosome prophylaxis and treatment.Due to praziquantel's low toxicity to eggs, it is not effective in preventing the progression of liver fibrosis caused by schistosomiasis infection According to pharmacological monographs, current literature, and experimental studies, metabolites or plant extracts have the potential to treat schistosome-induced liver fibrosis.Their therapeutic mechanisms are characterized by 1) reduction of inflammation, 2) reduction of the granulomatous response, 3) reduction of oxidative stress, and 4) reduction of egg loading (Figure 1).There is considerable evidence that therapeutic efficiency is improved by combining metabolites/ plant extracts with praziquantel, or with nanocarriers, or by using liposomes.Previous basic and clinical studies have shown good safety and tolerability for metabolic substances like resveratrol and chlorogenic acid, but adverse effects cannot be excluded due to experimental variability, inter-individual variability, and the lack of clinical trial reports.Furthermore, studies on the above metabolites and botanicals for the treatment of hepatic fibrosis due to schistosomiasis have been limited to basic research and no Pharmacologic mechanisms of natural products against schistosomiasis-associated liver fibrosis.
Frontiers in Pharmacology frontiersin.orgclinical studies have been reported.In conclusion, the protective role of natural products in the treatment of liver fibrosis in schistosomiasis needs to be confirmed by more standardized cellular studies, and supported by in vivo data from animal studies, which if promising should be escalated to randomized controlled clinical trials in humans.

TABLE 1
(Li et al., 2017) for schistosomiasis-associated liver fibrosis.reducedhepaticfibrosisbydecreasingtheexpression of pro-fibrotic factors (e.g., IL-13, IL-13 receptor α1, IL-4 receptor α), as well as by decreasing the expression of PPARγ, KLF4, SOCS1, and p-STAT6, and by inhibiting the polarization of M2 macrophage in Schistosoma hepatic tissues in mice(Du et al., 2016).Li et al.showed that Corilagin (39-157 μM, 24 h) significantly inhibited downstream fibrotic factors by interfering with the binding of IL-13 to IL-13Rα1 in Ana-1 cells(Li et al., 2017).Yang et al.found that treatment of schistosome mice with 20 mg/kg Corilagin reduced the number of liver eggs and effectively protected against liver fibrosis by inhibiting miR21 regulation of Smad7 and Smad1/2 phosphorylation (Yang significantly