## 3 Results
### Mechanism of Action of Harpagophytum Extracts on FLSs  
In order to assay the effects of the different extracts on cannabinoid receptor expression, they were added to cell culture medium at a concentration of 0.1 mg/mL for 24 h, then, the mRNA expression level of CB1 and CB2 receptors was analyzed. The HPE\({}_{\text{H2O}}\) and HPE\({}_{\text{DMSO}}\) and to a lesser extent, HPE\({}_{\text{EOH50}}\), were able to increase the CB2 mRNA expression level, whereas HPE\({}_{\text{EOH100}}\) did not show any effect (Figure 3). CB1 receptor mRNA expression level was increased by HPE\({}_{\text{H2O}}\) and HPE\({}_{\text{DMSO}}\) and to a lesser extent by HPE\({}_{\text{EOH100}}\), whereas it was decreased by HPE\({}_{\text{EOH50}}\) (Figure 3). Figure 1: Immunohistochemical analysis of synovial membranes from OA (osteoarthritis) and non-OA patients. Left panel: Slices were stained with anti-CB1 and anti-CB2 receptor antibodies. Right panel: Slices were stained with anti-PI-PLC \(\beta\)2 and anti-PI-PLC \(\beta\)3 antibodies. Slides were counterstained with hematoxylin and mounted with permanent mounting media. This figure shows representative images of different experiments (n = 5 non-OA and n = 6 OA). Considering that only CB2 receptors are associated with inflammation and pain in peripheral tissues, we verified whether the CB2 receptors also increased at the protein level by immunofluorescence staining. FLSs were treated with 0.1 mg/mL extracts for 24 and 48 h, then, the cells were stained with antibody anti-CB2. We found that \(\text{HPE}_{\text{H2O}}\) and \(\text{HPE}_{\text{DMSO}}\) extracts were able to stimulate the exposure in membrane of CB2 receptors, both at 24 and 48 h, whereas \(\text{HPE}_{\text{EOH50}}\) and \(\text{HPE}_{\text{EOH100}}\) did not stimulate CB2 receptor expression at any analyzed time (Figure 4 and Figure S1). Taking into account that the expression of PI-PLC 62 was increased in synovial membranes from OA patients, we verified whether the HPE extracts were able to reduce the production of this phospholipase. \(\text{HPE}_{\text{H2O}}\)  
Figure 3: Effects of all HPE extracts on CB1 and CB2 mRNA expression level in human primary FLSs. After 24 h treatment with 0.1 mg/mL of _Harapoylutum proclumbens_ root extract (HPE) dissolved in deionized water (\(\text{HPE}_{\text{H2O}}\)), DMSO (\(\text{HPE}_{\text{DMSO}}\)), 100% \(v\)/\(v\) EtOH (\(\text{HPE}_{\text{EOH100}}\)), and 50% \(v\)/\(v\) EtOH (\(\text{HPE}_{\text{EOH50}}\)), cells were harvested and mRNA was extracted and analyzed by RT-PCR. CB1 and CB2 receptor mRNA levels were reported as relative mRNA expression level with respect to 185 mRNA (\(2^{-\Delta\text{AC1}}\) method). Results are expressed as mean \(\pm\) S.E.M. of data obtained by three different experiments. Statistical significance was * \(p<0.05\); ** \(p<0.01\)**. Figure 2: Characterization of human primary synovicocytes (FLSs) and analysis of cell viability. (**A**) Human primary FLSs, isolated by synovial membranes and cultured in vitro, were stained with anti-vimentin primary antibody and with Alexa Fluor 488 (green) secondary antibody. (**B**) Cell viability was assessed by the MTS colorimetric method, and FLSs were treated with three concentrations, 1, 0.5, and 0.1 mg/mL of _Harapoylutum proclumbens_ root extract (HPE) dissolved in deionized water (\(\text{HPE}_{\text{H2O}}\)), DMSO (\(\text{HPE}_{\text{DMSO}}\)), 100% \(v\)/\(v\) EtOH (\(\text{HPE}_{\text{EOH100}}\)), and 50% \(v\)/\(v\) EtOH (\(\text{HPE}_{\text{EOH50}}\)), for 24, 48, and 72 h. Cell viability of treated samples was normalized to the untreated cells, which is reported as 100% and represented by a horizontal line. and \(\rm{HPE_{DMSO}}\) were able to inhibit the expression of PI-PLC \(\beta\)2, whereas \(\rm{HPE_{EOH50}}\) and \(\rm{HPE_{EOH100}}\) did not show effects (Figure 4). Figure 4: Effects of all HPE extracts on CB2 receptor and PI-PLC \(\beta\)2 protein production.