Фитотерапия боль в нижней части спины




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Discussion


Traditionally, H. procumbens has been used as an herbal medicine for a variety of conditions, and currently it is mainly used as an anti-inflammatory agent and as an analgesic [21]. In addition, it has been traditionally (but anecdotally) used as a modulator of appetite [43]. However, scientific studies evaluating the effect on food intake are lacking. In this paper we show, to our knowledge for the first time, the ability of H. procumbens root extract to modulate the GHS-R1a receptor, which is a key receptor implicated in appetite stimulation following activation by its agonist ghrelin. The H. procumbens extract was able to potently stimulate an intracellular calcium influx in vitro. In addition, the H. procumbens root extract-mediated GHS-R1a receptor modulation was enhanced in response to GHS-R1a receptor sensitization following pre-treatment with the inverse agonist peptide, [D-Arg1, D-Phe5, D-Trp7,9, Leu11]-substance P (SP). It was shown that the H. procumbens root extract-mediated GHS-R1a receptor response is not due to the presence of the main iridoid glycoside harpagoside, contained within the plant, suggesting that other compounds in the extract are responsible for the interaction with the GHS-R1a receptor.

We also analysed the effect of H. procumbens extract on GHS-R1a receptor internalization into endosomes, which is a characteristic of full receptor activation and subsequently follows receptor desensitization [41]. Both desensitization and internalization processes provide essential physiological “feedback” mechanisms that protect against both acute and chronic overstimulation of receptors [44], [45]. However, no internalization of the receptor was observed following H. procumbens root extract exposure suggesting that this extract does not act as a full GHS-R1a receptor agonist. Inverse agonists, like SP have been shown to decrease constitutive activity leading to enhanced ligand-mediated calcium signalling [38], [39], which is confirmed in this study as H. procumbens-mediated calcium increase through the GHS-R1a receptor is increased following pre-treatment with SP. However, H. procumbens root extract did not increase GHS-R1a receptor expression on the membrane like SP did and it is thus unlikely to act in a similar matter as an inverse agonist.

Receptors are phosphorylated by G-protein coupled receptor kinases (GRK) following agonist-mediated activation and this process activates proteins involved in G protein-coupled receptors (GPCR) internalization [44], [45], such as β-arrestin, which is the most widely standard adaptor for GPCR endocytosis [46]. Indeed, GHSR-1a receptor stimulation by the endogenous ligand ghrelin induces β-arrestin recruitment and activates the mitogen-activated protein kinase (MAPK) pathway (for review see [47], [48]). Perhaps, β-arrestin recruitment is not mediated by H. procumbens-mediated GHS-R1a receptor activation, as β-arrestin independent recruitment has also been demonstrated, which warrants further investigations. Thus, we show that the H. procumbens-mediated intracellular calcium signalling alone is not sufficient to promote GHS-R1a receptor internalization and full receptor activation. Interestingly, a significant dose-dependent decrease in food intake was observed following intraperitoneal administration of H. procumbens root extract in ad libitum fed mice as well as a decrease in intake in food restricted mice. The major significant appetite effect of H. procumbens occurs within the first 2 hours of administration (Figure 5, 6).6). This early effect on appetite is in line with the orexigenic effects of the endogenous GHS-R1a receptor ligand, ghrelin, which are also only observed within the first 2 hours of administration [34].

We hypothesize that the H. procumbens root extract interacts with the GHS-R1a receptor, as elicited by increased intracellular calcium influx, but does not lead to subsequent GHS-R1a receptor internalization and, therefore, does not act as full GHS-R1a receptor agonist. This may suggest that the GHS-R1a receptor internalization is required for further down-stream orexigenic effect in vivo. It is indeed tempting to speculate that the orexigenic effects of the endogenous ligand ghrelin are dependent on GHS-R1a receptor internalization, which occurs immediately following calcium signalling. Moreover, we suggest that modulation of the GHS-R1a receptor by the H. procumbens extract may decrease the availability of the receptor to the orexigenic effects of ghrelin, which may explain the observed anorexigenic effects.

Further investigations are needed to clarify the precise GHS-R1a receptor-mediated intracellular signal transduction pathways and correlate these to physiological behaviours. A recent study has shown that GHS-R1a receptor knock-down, inverse agonism, or desensitization can exert the same biological effect under certain circumstances [49]. However, in relation to food intake, GHS-R1a receptor inverse agonism has been reported to reduce appetite and body weight gain [49] in contrast to the orexigenic effects of ghrelin. Interestingly, biased agonism has also been reported for GPCRs modulation leading to different active receptor conformations adding to the complexity of GHS-R1a receptor signalling (for review [48]). Hence, different intracellular signal transduction pathways may be triggered by ghrelin, inverse agonists and H. procumbens extract.

Furthermore, we cannot rule out that the H. procumbens root extract interacts with other receptors implicated in satiety, such as the serotonin receptors (5-HT1B, 5-HT2C and 5-HT6), cholecystokinin receptor (CKK-A) and glucagon-like peptide-1 receptor (GLP-1R), suppressing appetite. However, to our knowledge no such interactions have been reported to date. Future studies using GHS-R1a receptor antagonists or GHS-R1a knock-out mice may be able to further delineate the molecular mechanism of the GHS-R1a receptor-dependent anorexigenic effects of H. procumbens. Finally, it would also be interesting to investigate if the H. procumbens-mediated anorexigenic effect is maintained following oral administration and if the bioactive has proteolytic stability in transit.

Following compositional analysis, the most abundant compounds in the dried H. procumbens root powder were demonstrated as carbohydrates. Among these compounds, the most potential active constituent of the extract are iridoids glycosides (for review see [20]). In this study we show that harpagoside, previously demonstrated as the main iroid glycoside in H. procumbens [50], does not have any GHS-R1a activating potential. Therefore, harpagoside is not implicated in the interaction between H. procumbens extract and the GHS-R1a receptor. However, others iridoids glycosides may be implicated. In addition, the dried H. procumbens root was also rich in fibre. Several studies have shown that consumption of diets rich in fibre lead to beneficial anti-obesity effects such as increased satiety, reduced hunger, reduced food intake, and body weight loss (for review see [51]). Fibre exerts these anti-obesity effects by acting in the gastrointestinal tract through different mechanisms such as increasing gastric distension, delaying gastric emptying, digestion and absorption of nutrients, increasing insulin and glycemic responses, affecting gut hormones secretion such as GLP-1, peptide YY and neurotensin, reducing the absorption of fat and increasing the fecal energy excretion (for review see [51]). Therefore, fibre may also be potentially implicated in the decreased food intake observed in vivo by affecting gastrointestinal digestion process. However, further analyses are needed to investigate a possible interaction of fibre with the ghrelin receptor, which may be also implicated in the anorexigenic effect of H. procumbens. Future studies are needed to identify the specific bioactive responsible for the appetite suppressant effects of H. procumbens. Nevertheless, as demonstrated in this study, the crude H. procumbens extract has potent anorexigenic effects, which would be sufficient to be utilized as a natural anti-obesity treatment in its un-purified form. This significantly contributes to its potential commercial application.

We conclude that H. procumbens root extract is a novel source for potent anti-obesity bioactives with GHS-R1a mediated appetite suppressant effects. Therefore, H. procumbens root extract may represent a possible natural alternative which may be safer and more attractive compared to current pharmacological drugs, which are often associated with several side effects. Hence, the identification of the GHS-R1a receptor modulating bioactive from H. procumbens is poised to have important therapeutic potential in obesity and obesity related diseases.



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