Characteristics of ginsenoside Rd-induced effects on rat intestinal contractility with irritable bowel syndrome

Abstract Irritable bowel syndrome (IBS) is a very common refractory disease. Its exact pathophysiological mechanism is still unclear. Despite the availability of plentiful drugs to control IBS, most patients do not respond well. Ginsenoside Rd is one of the major active components of Panax ginseng, which has been verified to produce various pharmacological actions. However, the role of ginsenoside Rd in modulating smooth muscle contractility is still undefined. The aim of this study is to investigate the effects of ginsenoside Rd on intestinal contractility and related mechanisms in IBS. Graphical Abstract


Introduction
Irritable bowel syndrome (IBS) is the most common dysfunction of gastrointestinal system, characterised by irregular bowel movements, abdominal discomfort, and stool consistency (Chen et al. 2021). Based on clinical manifestations, IBS is subtyped based on the predominant bowel habit: diarrhea (IBS-D), constipation (IBS-C), mixed diarrhea and constipation (IBS-M), and unspecified (IBS-U) (Nordin et al. 2022). The pathogenesis of IBS is intricate, which may be related to abnormal colonic motility, hypervigilance, and symptom-related anxiety. Substantial progresses have been made in comprehending its intricate pathophysiology, leading to its classification as a dysfunction of gut-brain interaction (Vasant et al. 2021). The treatment strategies for IBS are focused on administrating intestinal motility function and decreasing intestinal sensitivity. The costs associated with IBS are estimated to be reached billion US dollars in the USA, and 123 billion Chinese RMB in China (Ford et al. 2020). Although the availability of numerous drugs to cure IBS, most patients respond unwell (Chey et al. 2015). Some popular drugs for IBS, such as anticholinergic medications and antispasmodic, do not ameliorate all symptoms and may produce adverse reactions like abdominal distension and constipation (Chen et al. 2021). Treatment for IBS using Chinese medicine has attracted more and more attention as a result of the advantages of cost-effectiveness, few side effects, convenience, and simplicity.
Ginsenosides are the major active components of Panax ginseng (Christensen 2009), which have plentiful pharmacologic effects like anti-tumorigenic (Surh et al. 2001), immunomodulatory, anti-stress effects (Leung and Wong 2010), protection of the cardiovascular system (Shi et al. 2011), neuroprotective (Gong et al. 2011), antiinflammatory , and antidiabetic (Kim et al. 2011). Recently ginsenosides exerted bidirectional modulation on the contractility of isolated jejunal segment (Chen et al. 2014). The results showed the potential clinical significance of ginsenosides for alternating IBS. Further research is needed to clarify which components for ginsenosides-induced bidirectional modulation.
Although the content of ginsenoside Rd is relatively low in ginsenosides extracted from Panax ginseng (Chen et al. 2022), Ginsenoside Rb 1 , Rb 2 , and Rc which have the similar structure can be converted into Rd by microorganisms or enzymes. Ginsenoside Rd reveals the best in pharmacological activity than that of other ginsenosides (Rb 1 , Rb 2 , Rc), because it has higher ability across the cell membrane (Zhang et al. 2021). Thus, it is that ginsenoside Rd might be the candidate component for ginsenosides-induced bidirectional modulation to control IBS.

Characteristics of ginsenoside Rd-induced effects on contractility of jejunal segment
The isolated jejunum segment had stable spontaneous contraction ginsenoside Rdinduced effects on jejunal contractility under the normal contractile were shown by its dose-response relationship ( Figure S1). Ginsenoside Rd increased contractile amplitude of isolated jejunal segment in the concentration range of 2.5 $ 20 lmol/L (p < 0.05; n ¼ 6 tissues) and decreased contractile amplitude of isolated jejunal segment in the concentration range of 40 $ 80 lmol/L (p < 0.05; n ¼ 6 tissues).

Ginsenoside Rd-induced bidirectional modulation on jejunal contractility
To assess ginsenoside Rd-induced effects in different conditions, 6 low and 6 high contractile states of jejunal segment were set up and used in the research. The contractility of isolated jejunal segment in both low and high contractile was significant difference from that in normal contractile. Ginsenoside Rd exerted bidirectional modulation, that is, stimulatory effects on the contractility of isolated jejunal segment in all 6 low contractile states ( Figure S2-1) and inhibitory effects on the contractility of isolated jejunal segment in all 6 high contractile states ( Figure S2-2). Ginsenoside Rd at the concentration of 10 lmol/L.

The role of enteric nervous system in ginsenoside Rd-induced bidirectional modulation
To assess the role of enteric nervous system (ENS) in ginsenoside Rd-induced bidirectional modulation, in the presence of TTX, only inhibitory effects exerted by ginsenoside Rd on the contractility of isolated jejunal segment were observed in the normal contractile state, low contractile state and high contractile state, respectively. ( Figure S3).

The role of Ca 2þ in ginsenoside Rd-induced bidirectional modulation
Ginsenoside Rd (5 $ 80 lmol/L), did not exert further modulation on the contractility of isolated jejunal segment under pre-incubated with verapamil (1 mmol/L) or Ca 2þ free assay condition ( Figure S4).

The role of interstitial cells of Cajal in ginsenoside Rd-induced bidirectional modulation
The results showed that imatinib (5 lmol/L) itself doesn't have different impact on the effect of the jejunal segment under the normal contractile state, low contractile state and high contractile state. Since, in the presence of imatinib, ginsenoside Rd (10 lmol/ L)-induced bidirectional modulation whereas in the absence, i.e. stimulatory effects on the jejunal segment in low contractile state and inhibitory effects in high contractile state were abolished ( Figure S5).

Targets related to ginsenoside Rd-induced bidirectional modulation
Atropine obstructed the stimulatory effects of ginsenoside Rd on the contractility of isolated jejunal segment in low contractile state ( Figure S6 and Table S1). Diphenhydramine did not affect ginsenoside Rd-induced stimulatory effect on jejunal contractility in low contractile state ( Figure S6 and Table S1); phentolamine, propranolol, and Nomega-Nitro-L-arginine (L-NNA) blocked ginsenoside Rd-induced inhibitory effects on the contractility of isolated jejunal segment in high contractile state, respectively ( Figure S6 and Table S1).
Constipation or diarrhoea is closely associated with the disorder of intestinal contraction (Sjolund and Ekman 1987). It has been uncovered that the symptoms of the high intestinal contractility are manifested as diarrhoea, and the symptoms of low contractile are manifested as constipation (Gershon 2004;Seidl et al. 2009). There was dose dependent effect of ginsenoside Rd on isolated jejunal segment in normal contractile state, namely, ginsenoside Rd increased and decreased the contractility of isolated jejunal segment in the dose range of 2.5 $ 20 lmolÁL À 1 and 40 $ 80 lmolÁL À 1 , respectively. Ginsenoside Rd in the fixed concentration of 10 lmolÁL À 1 induced bidirectional modulation on the contractility of isolated jejunal segment, that is, stimulatory effects on all 6 low contractile states, and inhibitory effects on all 6 high contractile states. In the absence of stimulatory or inhibitory effects of ginsenoside Rd on isolated jejunal segment were observed when assayed in pretreated with the verapamil (Ca 2þ channel blocker) or a Ca 2þ free buffer, indicating that the effects of ginsenoside Rd on jejunum were backed by the influx of extracellular Ca 2þ . The stimulatory effects of ginsenoside Rd on the contractility of isolated jejunal segment in low contractile states were connected with M-receptor-linked stimulation, because of they were dependant on the atropine (M receptor antagonist). The inhibitory effects of ginsenoside Rd on the contractility of isolated jejunal segment in high contractile states were connected with adrenergic aand b-receptors, as well as NO-synthase activity-related modulation, since phentolamine, propranolol, and L-NNA blocked the inhibitory effects of ginsenoside Rd on the contractility of isolated jejunal segment in high contractile states, respectively.

Effects of ginsenoside Rd-induced bidirectional modulation on myosinphosphorylation
As shown in Figure S7-1, the phosphorylation of 20 kDa myosin light chain  in IBS-C group and IBS-D group was remarkably decreased and increased respectively in comparison with that in the normal control group (p < 0.01, n ¼ 4 rats). Ginsenoside Rd (30 mg/kg once daily for 14 consecutive days) remarkably increased p-MLC 20 in ginsenoside Rd-treated IBS-C group and remarkably decreased p-MLC 20 in ginsenoside Rd-treated IBS-D group in comparison with those of untreated IBS-C and untreated IBS-D groups, respectively (p < 0.01, n ¼ 4 rats).
As shown in Figure S7-2, the protein content of myosin light chain kinase (MLCK) in IBS-C group was remarkably decreased in comparison with the normal control group, and the ginsenoside Rd-treated IBS-C group was remarkably increased compared with IBS-C group (p < 0.01, n ¼ 10 samples); the content of MLCK in the IBS-D group was remarkably increased in comparison with that in the normal control group, and the ginsenoside Rd-treated IBS-D group was remarkably decreased compared with IBS-D group (p < 0.01, n ¼ 10 samples).As shown in Figure S7-3, the effects of ginsenoside Rd on the expression of MLCK mRNA were close correlation with the effects of ginsenoside Rd on the content of MLCK.
IBS-C and IBS-D are two common intestinal types, which have inverse symptoms, but both are associated with the abnormal intestinal contraction in response to psychological and mental factors (Ohman and Simren 2007). The conventional medicine for IBS treatment aims to alleviates the common symptoms and thus adopts opposite therapies for constipation and diarrhoea because of the distinct symptoms. Smooth muscle relaxation and contraction are primarily modulated by dephosphorylation and phosphorylation of MLC 20 by myosin light chain phosphatase and MLCK, respectively (Jeong et al. 2011). In conformity to the modulation on intestinal contractility, the effects of ginsenoside Rd on myosin phosphorylation, the contents of MLCK, and the expression of MLCK mRNA in intestinal tract determined in low-high contractile states were also bidirectional, indicating that ginsenoside Rd-induced inhibitory and stimulatory effects are associated with the dephosphorylation and phosphorylation of MLC 20 , respectively.

Conclusions
Ginsenoside Rd-induced bidirectional modulation needs the presence of ENS, depends on the influx of extracellular Ca 2þ , relates to the cholinergic system while isolated jejunal segment is in low contractile states, and relates to the adrenergic system and NO relaxing mechanism while isolated jejunal segment is in high contractile states. The myosin light chain kinase related signaling pathway is also involved in ginsenoside-induced bidirectional modulation. Further study is required since it remains unclear as to the detailed mechanisms for ginsenoside Rd-induced bidirectional modulation, including how dozens of neurotransmitters in the jejunum are interrelated in various contractile states and how they are integrated in regulation. The results suggested the potential implication of ginsenoside Rd for alternating IBS. In short, our data showed that ginsenoside Rd improves disease symptoms of both rat constipation and diarrhoea in a bidirectional modulation, giving evidence of ginsenoside Rd as an potential drug in the treatment of IBS.