Phd in BIOCHEMISTRY

Thesis title: From root extract to antiarthritic facts: trip across a novel therapeutic approach to inflammation and pain

Osteoarthritis (OA) is a chronical-degenerative and inflammatory disease affecting joints and involving several cellular and molecular processes in different cell types, as chondrocytes, osteoblasts, synoviocytes and immune cells. In OA the cartilage, a specialized tissue that allows the sliding between the two joint heads, is degraded, and, in the joint, the bones have friction each other causing pain and their own remodeling. Synovial membrane cells secrete inflammatory mediators and a greater amount of synovial fluid, resulting in synovial membrane thickening and contributing to the joint damage. To date, there is no specific anti-OA therapy, and so painkillers, anti-inflammatory drugs and intra-articular injections of corticosteroids are administered. However, considering painkilling and anti-inflammatory drug contraindications, in addition to chondroprotectors that preserve cartilage from degeneration, nutraceuticals are often introduced in therapies for their efficacy and less side effects than traditional drugs. Among them, Harpagophytum procumbens, known as Devil’s claw, is one of the most used herbs as an anti-OA remedy. Its secondary roots contain several bioactive compounds able to relieve patient symptoms decreasing inflammation and joint pain. In order to understand its activity on pain and inflammatory pathways, in the present study, a H. procumbens root extract (HPE) was characterized for its phytochemical composition and effects on Fibroblast- like synoviocytes (FLSs) from OA patients. The dry HPE was solubilized in different solvents, deionized water (HPEH2O), DMSO (HPEDMSO), 100% v/v ethanol (HPEEtOH100), and 50% v/v ethanol (HPEEtOH50), chosen based on their biocompatibility and ability to dissolve different classes of phytochemical compounds. Preliminary phytochemical analyses have shown that the highest polyphenol levels were found in HPEDMSO and HPEEtOH50, whereas different volatile bioactive compounds, mainly β-caryophyllene and eugenol, were detected in all the extracts except for HPEH2O. Since these compounds have been described to mitigate pain through an agonism on endocannabinoid type 2 (CB2) receptors and considering the involvement of CB2 in OA pathogenesis demonstrated through our histochemical analyses, it was decided to analyse the HPE analgesic effect on OA FLSs. However, even if both HPEH2O and HPEDMSO were able to enhance CB2 receptor expression, only the latter has been used for subsequent experiments. Considering the CB2 receptor association with Gi protein in cell membrane, the HPEDMSO effectiveness to affect CB2 pathways was studied by analysing the cAMP modulation, the protein kinase A (PKA) and Extra-regulated kinase (ERK) activation and the correlated matrix metalloproteinase (MMP) production. In line with what expected, HPEDMSO was able to inhibit cAMP production and PKA activation, also showing a reduction in ERK1/2 phosphorylation that surprisingly disagreed with the extract effect on PKA. Interestingly, although it is not due to CB2 receptor stimulation, the effect on the MAPK pathway generates a decrease in some extracellular matrix degrading enzyme and pro-inflammatory interleukin expression, suggesting the HPE antiarthritic role. Moreover, to clarify the effects of bioactive constituents and the possible interactions occurring in the phytocomplex, harpagoside, the H. procumbens root extract biomarker, and the main volatile compounds detected at the phytochemical analysis, have been studied in comparison to the whole extract. It was observed that β-caryophyllene, α-humulene, eugenol and harpagoside alone were always less effective than the H. procumbens whole extract and the Mix that contained all the individual compounds. However, the surprising result is that the mixture was also always less effective than HPEDMSO whole extract. This evidence suggested the existence of synergistic interactions between each analysed compound and other molecules not identified in phytochemical analyses that contribute to the entire extract bioactivity. In our most recent analyses, HPEDMSO has demonstrated a role in the inhibition of the phosphoinositide specific-phospholipase C (PI-PLC) γ1 mRNA splicing process. Sanger sequencing analysis revealed some retained introns in HPEDMSO treated FLSs. However, studies describing the H. procumbens ability to interfere with the splicing process were not available in scientific literature. On the other hand, the accurate analysis of the PI-PLC γ1 gene, only carried out by the Human and Vertebrate Analysis and Annotation (HAVANA) group, identified the intron retention phenomenon in PI-PLC γ1 gene. Our results were perfectly in line with what described by the HAVANA group. However, during our analysis we highlighted a lack of HPEDMSO effect on intron retention phenomenon in some FLS samples from different OA patients. This inter-individual variability led us to consider more carefully some data obtained in our previous experiments. Surprisingly, it was shown that HPEDMSO was ineffective in inhibiting MMP gene expression in those samples where the intron retention phenomenon in PI-PLC γ1 mRNA was absent. This result suggested that the decrease of PI-PLC γ1 protein, due to lack of splicing, could be associated with the decrease of MMP production.