Background
Schistosomiasis is the most prevalent and debilitating neglected diseases in the tropics and subtropics. It is associated with severe morbidity and mortality. Approximately 7 weeks after the initial transcutaneous infection, schistosomes migrate to the host hepatic portal system and egg production starts in the tributaries of the inferior mesenteric vein [
1]. Adult
Schistosoma mansoni worms reside in the mesenteric veins for years where they lay eggs and release antigens, which eventually lead to death. The antigen-laden eggs are taken with the blood flow and embolize in the liver, causing granulomatous hepatitis leading to fibrosis and portal hypertension [
2]. In Latin America, Africa, and Asia, schistosomiasis is one of the most important cause of noncirrhotic portal hypertension [
3].
S. mansoni eggs induced a granulomatous reaction associated with an overproduction of reactive oxygen and nitrogen species, as well as an impairment of the antioxidant defence [
4‐
8]. During
S. mansoni life cycle in humans, a part of the eggs reaches the terminal ileum and the colon to be excreted along with the feces, while the other part remains entrapped within the gut wall. In that location, antigenic substances released by the eggs initiate granulomatous inflammation, resulting in intestinal lesions [
1]. Eighty-five percent of
S. mansoni-infected individuals suffer from motility-related gastrointestinal symptoms such as diarrhea, anorexia, nausea, abdominal cramps and constipation [
2,
9,
10]. These clinical manifestations may result from gastrointestinal neuromuscular dysfunction induced by the inflammatory cells infiltration in the gut wall [
1]. The effect of granulomatous inflammation on gastrointestinal motility in
S. mansoni infection has been demonstrated. Gastrointestinal transit was inhibited during chronic infection and was associated with inflammation of the intestine wall and with an increase of in vitro contractility of longitudinal muscle strips of the ileum [
1,
2,
11,
12]. A schistosomicidal treatment that could prevent or suppress the granulomatous inflammation, could, therefore, avert the development of severe disease [
13].
For two decades, praziquantel has remained the only drug against all human schistosome species. Poor cure rates and reduced susceptibility of this drug among some isolates of
S. mansoni has been demonstrated [
14]. This raised concerns about the search and development of complementary and/or alternative new schistosomicidal drugs that are both effective and safe. The value of many plant species used in traditional medicine to treat schistosomiasis is increasingly being recognized.
Sida pilosa Retz (Malvaceae), is a creeping plant founded mainly on the outskirts of dwelling areas and on wastelands. In Cameroon, the macerate of the plant aerial parts is used for the treatment of intestinal helminthiasis [
15]. Pharmacological and toxicological studies have demonstrated the schistosomicidal activity and the safety of
S. pilosa aqueous extract. Phytochemical screening has revealed the presence of alkaloids, phenols, flavonoids, tannins, and terpenoids in the extract [
16]. Fractionation of
S. pilosa aqueous extract was performed and the HPLC-MS analysis of its
n-butanol fraction revealed the presence of two indoloquinoline alkaloids. Moreover, this fraction displayed good in vitro activity against
S. mansoni [
17]. However, in vivo studies on the evaluation of the schistosomicidal, antioxidant, anti-inflammatory and anti-fibrotic activities of
S. pilosa aqueous extract and its
n-butanol fraction has demonstrated that the aqueous extract was more active than the fraction [
7]. The spasmogenic effect of
Sida veronicaefolia (syn.
Sida pilosa) on ileal and uterine smooth muscle has been also reported [
18,
19]. The present study was performed to evaluate the effect of
S. pilosa aqueous extract on the granulomatous inflammation in the liver and the intestine by histomorphometry as well as on the gastrointestinal transit after
S. mansoni infection. To verify the hypothesis that
S. pilosa aqueous extract could contribute to maintaining a normal gastrointestinal transit, its effect on the contractile activity of the ileum was investigated.
Discussion
Sida pilosa aqueous extract (SpAE) have been shown to be effective against
Schistosoma mansoni infection in the murine model through the reduction of hepatosplenomegaly, worm burden and egg burden in the feces and the liver [
16]. Most recently, antioxidant, anti-inflammatory and anti-fibrotic activities of SpAE in
S. mansoni-infected mice were demonstrated through the evaluation of some liver biomarkers [
7]. Based on histology supplemented with morphometry, this study has assessed the effect of SpAE on the granulomatous inflammation induced by
S. mansoni infection in the liver and the intestine. Gastrointestinal motor activity was also evaluated. We demonstrated the presence of granulomas surrounding entrapped
S. mansoni eggs in the liver of mice after 10 weeks of infection. These granulomas consisted mainly of collagen fibers and scarce inflammatory cells. In the ileum, mucosal inflammation and granulomas consisted mainly of macrophages and eosinophils surrounded by lymphocytes were present. An increase of the ileal muscular layer thickness was also observed. This was linked to the infiltration of inflammatory cells in the mucosa and submucosa. Described by Lenzi et al. [
31] as an organoid, dynamic and hybrid structure formed by eosinophils, neutrophils, lymphocytes, macrophages, epithelioid cells, giant cells, mast cells, reticular fibres and fibroblasts surrounding schistosome eggs trapped in an organ, the schistosomal granuloma is commonly present in the liver, the intestine, the spleen and the lung of
S.mansoni-infected animals [
1,
2,
13,
21,
32]. The increase of intestinal wall thickness after
S. mansoni infection has also been described in previous studies [
1,
2,
11].
In the current study, morphometric analyses of the liver and the ileum demonstrated that
S. mansoni infection considerably increases the number of granulomas in the liver and the intestine, their volume in the liver, as well as the ileal muscular layer thickness. The presence of numerous and voluminous granulomas in the liver and the intestine of
S. mansoni-infected mice probably explain the enlargement of these organs. The regression of hepatosplenomegaly and intestine enlargement after SpAE treatment could follow the reduction of
S. mansoni egg load in the liver, the spleen, and the intestine. Jatsa et al. [
7,
16] have in fact demonstrated the schistosomicidal activity of SpAE through the reduction of the liver and intestine egg burden. Since the inflammatory reaction leading to the formation of a granuloma is initiated by schistosome eggs trapped in organs as liver and intestine, the consequence of the reduction of egg load in those organs could be the reduction of the recruitment and migration of inflammatory cells around schistosome eggs, and therefore the granulomatous reaction. In
S. mansoni infection, if granuloma formation could be prevented or suppressed, the development of severe disease might be averted [
13]. Data from the present study showed that treatment of
S. mansoni-infected mice with SpAE significantly reduces granuloma number in the liver and the ileum by 52.82% and 52.79% respectively, as well as granuloma volume in the liver by 48.76%. The thickness of the ileal muscular layer was slightly but significantly reduced by 10.52%. Reduction of granuloma number and/or size has been previously reported by others authors after treating
S. mansoni-infected mice with curcumin [
13],
Zingiber officinale rhizomes [
33], berberine [
4],
Ceratonia siliqua pod [
8], selenium nanoparticles [
5] or vaccination of mice with the tegumental antigen of
S. mansoni adult worms prior to infection [
34]. The formation and the development of hepatic granuloma are intimately linked to inflammatory cells infiltration in the liver. The reduction of these cells infiltration would, therefore, induce a reduction of hepatic granuloma. It has been proved that SpAE reduces eosinophils’ and neutrophils’ infiltration through reduction of eosinophil peroxidase and myeloperoxidase activities [
7]. The reduction of the granuloma number and volume could also be attributed to the anti-inflammatory and antifibrotic properties of SpAE as recently demonstrated in a murine model of
S. mansoni infection [
7]. In this study, SpAE exacerbated anti-inflammatory activity through the restoration of ALT and AST activities in infected mice. ALT and AST are biomarkers of the integrity of hepatocytes. In
S. mansoni-infected mice, deposition of schistosome eggs in the liver induces granulomatous lesions which leads to the release of transaminases from the injured hepatic cells to the bloodstream. This granulomatous reaction is also associated with the generation of reactive oxygen species and the impairment of antioxidant defense [
7,
8,
13]. The reduction of the granuloma number and volume in the liver of infected-treated mice could be correlated to the normalization of transaminases activity in these mice. The antioxidant potential of SpAE could also contribute to the reduction of the granulomatous reaction. Results from this study shown that SpAE displayed significant antioxidant activity through the reduction of malondialdehyde concentration and the activation of antioxidants such as catalase, superoxide dismutase, and reduced glutathione. The in vitro and in vivo antioxidant potential of SpAE has been previously demonstrated and was correlated to the high content of total phenolic compounds of this extract [
7,
17]. Since reactive oxygen species act as both signaling molecules and mediators of inflammation, the reduction of their concentration in the liver will be in favor of the reduction of granulomatous inflammation.
During
S. mansoni infection, morphological alterations of the ileum might affect intestinal smooth muscle function, leading to a dysfunction of the gastrointestinal transit [
1,
2,
12]. In this study, when granulomas were present in the ileum of
S. mansoni-infected mice, a significant decrease of the small intestine length and a significant reduction of the gastrointestinal transit were recorded. The current study, in line with findings of previous reports [
1,
35] indicates that during
S. mansoni infection, gastrointestinal motility is substantially altered. The presence of mucosal inflammation of the ileum probably inhibits the propulsive activity of the normal gut, leading to a dysfunction of the intestinal peristalsis and thus a decrease of the gastrointestinal transit [
1]. Abdu [
12] has reported a significant variation of peristaltic pressure waves amplitude and intervals in a murine model of
S. mansoni infection. Clinical manifestations of intestinal schistosomiasis such as constipation could be the consequence of a dysfunction of the gastrointestinal motor system [
9,
10]. A normal gastrointestinal transit was however restored after the treatment of
S. mansoni-infected mice with SpAE which produced a prokinetic effect. This result could be attributed to the reduction of the granulomatous inflammation of the ileum which consequently suppresses the inhibition of the intestinal peristalsis and thus of the gastrointestinal transit. An increase of the gastrointestinal transit has been reported in the literature, where the flavonoid isoliquiritigenin isolated from the roots of
Glycyrrhiza glabra and
Terminalia chebula displayed contractile effect on isolated rat stomach fundus or ileum [
20,
36]. Another explanation for the restoration of the gastrointestinal transit could be the increase of the contractile activity of ileal muscles by SpAE. To verify this hypothesis, the effect of SpAE on the contractile activity of the ileum was studied.
Sida pilosa aqueous extract (SpAE) elicited a spasmogenic effect on the rat ileum by significantly increased, on a concentration-dependent manner the amplitude of spontaneous contractions (EC
50 = 4.49 mg/mL), but decreased the basal tone of spontaneous contractions (EC
50 = 17.35 mg/mL). In previous studies, the aqueous and ethanolic extracts of
Sida veronicaefolia (syn.
Sida pilosa), as well as sidaverin derived from
S. veronicaefolia, have been shown to exhibit a spasmogenic effect on ileal and uterine smooth muscle [
18,
19].
The gastrointestinal smooth muscles are spontaneously active with the periodic generation of slow waves whom the activity is initiated by the interstitial cells of Cajal distributed in the myenteric region [
37]. Slow waves propagate within the interstitial cells of Cajal network, reach the smooth muscle cells via gap junctions and initiate phasic contractions by activating Ca
2+ entry through L- type Ca
2+ channels. The gastrointestinal motility is an integrated process modulated by local and circulating neurohumoral substances [
37,
38]. In the present study, we investigated the mechanism underlying the action of SpAE on muscle contractions. The contractile effect of SpAE might involve muscarinic receptors, glutamate NMDA receptors or calcium influx into the muscle cell through voltage-gated Ca
2+ channels. Activation of muscarinic receptors on the gastrointestinal smooth muscle cells by releasing acetylcholine from excitatory neurons plays an important physiological role in the stimulation of spontaneous muscle contractions and atropine blocks all muscarinic receptor sites. The predominant muscarinic receptor subtypes present in intestinal smooth muscles are M2 and M3, and direct contraction of the intestinal smooth muscle is mediated via the M3 subtype [
29]. The failure of atropine (1 μM) to reduce the SpAE-induced contractions may indicate that the action of the extract is not through M3 muscarinic receptors. Glutamate evoked contractions of the longitudinal muscle/myenteric plexus (LMMP) preparation by acting on N-Methyl-D-aspartate (NMDA) receptors. Other agonists at the NMDA recognition site, but not quisqualate or kainate, also contracted the LMMP, and glutamate-evoked contractions are competitively inhibited by selective NMDA receptor antagonists. Evidence in the literature demonstrates that tramadol antagonizes glutamate NMDA receptors [
39]. In the present study, tramadol (50 μM) did not inhibit the spasmogenic effect of SpAE on ileal muscles contractions, suggesting that the action of SpAE is not through glutamate NMDA receptors. Since it has been demonstrated that tramadol also suppresses the activity of M3 muscarinic receptors [
39], the failure of tramadol to inhibit SpAE-induced contractions confirmed the non-involvement of the cholinergic system in the mechanism of action of SpAE. In smooth muscle cells, the primary stimulus for contraction is the increase in cytoplasmic calcium concentration. This increase is generally the result of both the release of intracellular Ca
2+ from sarcoplasmic reticulum stores and the influx of extracellular Ca
2+. The main pathway for extracellular Ca
2+ entry into intestinal smooth muscle cells is through L-type voltage-dependent Ca
2+ channels [
28,
29,
38]. Blockade of the L-type Ca
2+ channels using verapamil or another Ca
2+ channel blocker abolishes or markedly reduces the contractile activity of intestinal muscle strips [
29]. Results from this study showed that ileal muscle contractions induced by SpAE were drastically reduced by verapamil (0.05 μM), indicating that the action of SpAE on gastrointestinal motility is mediated by Ca
2+ influx through L-type Ca
2+ channels. Furthermore, SpAE-induced ileal muscle contractions were significantly reduced in a Ca
2+-free medium with EDTA 1 mM (a calcium chelator), suggesting that SpAE mobilizes Ca
2+ from sarcoplasmic reticulum stores.
The action of SpAE on the gastrointestinal motility is thus mediated via the mobilization of intracellular and extracellular calcium. The spasmogenic effect of SpAE may explain the prokinetic effect displayed in vivo. Chen et al. [
36] have in fact demonstrated that the increase of the gastrointestinal transit of rats who have received the flavonoid isoliquiritigenin was correlated to the spasmogenic effect of this compound. The role of bioactive compounds such as alkaloids, phenols, and terpenoids in promoting contractile effect on rabbit ileum and jejunum has been demonstrated [
40]. It has also been reported that tannins and flavonoids have an inhibitory effect on intestinal smooth muscles contractility [
41]. The dual effect of SpAE, increasing the amplitude of spontaneous contractions but decreasing their basal tone, might, therefore, be explained by the presence of alkaloids, phenols, triterpenes, and flavonoids in SpAE [
7,
16].