key: cord-0429827-zifjldsj authors: Arokiaraj, Mark Christopher; Menesson, Eric title: Rose Extract Treatment on the CD4+ T lymphocytes date: 2021-04-06 journal: bioRxiv DOI: 10.1101/2021.04.06.438604 sha: 5e7d239f4a54fa94a3f501d179509f4057eb91dd doc_id: 429827 cord_uid: zifjldsj Background and Aims To study the effect of rose extract on CD4+T lymphocytes, and assess the cytokines response after cell treatment. In our previous study on endothelial cells, the rose extract reduced the secretion of inflammatory markers significantly. Methods The red rose extract used in this study was prepared and stored until use at -20°C. T cells were seeded in 96-well plates at 313500 cells/well in 100μl of cell culture medium in duplicate, one half of the wells were used for biomarkers screening in the culture medium, and the other half for cytotoxicity assay. 24h after plating, the cells were treated in duplicate with 100μl of red rose extract diluted at 0.5%, 0.1%, 0.05%, 0.01% and 0.005% (v/v) in cell culture medium or with culture medium only as control for 72h. Some other wells were for untreated cells, and cells treated with rose extract at 0.005% for 48h incubation time. After 48h and 72h, the corresponding wells were used for the cytotoxicity assay and from the duplicate wells, the cell culture media were collected and stored at -80°C until the biomarkers screening assay. Results Cytotoxicity assay revealed insignificant changes. IFN-gamma, MCP-1, GRO, RANTES and TIMP, Angiopoietin 1 and MMP-9 were elevated. Except MMP-9 which had fold changes >2 other cytokines were minimally elevated at various concentrations and timing of rose extract treatment. None of the cytokines were less than 0.8-fold. Conclusions Unlike in the endothelial cells, there is mild elevation in few inflammatory markers on T lymphocytes treatment by rose extract. Further studies need to be performed to estimate the clinical relevance. CoV-2 spike protein on target ACE-2 receptors. 2 Currently existing most anti-inflammatory agents like steroids, 3 tacrolimus, 4 MMF 5,6 etc., reduce lymphocyte cell proliferation, and decrease inflammation. Monoclonal antibodies like rituximab, 7-9 infliximab 10 etc., are also associated with various side effects. Rituximab, which is widely used in various lymphoproliferative disorders and autoimmune disorders has anti-CD20 and also increases IL10. The red rose extract used in this study was prepared for our previous study 1, 11 ). This kit uses WST-8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt) which produces a water-soluble formazan dye upon bio-reduction in the presence of an electron carrier, 1-Methoxy PMS. WST-8 (10% of the medium volume in wells) was added to the media for 4h at 37°C. During the incubation time, it is bio-reduced by cellular dehydrogenases to an orange formazan product that is soluble in culture medium. Then, the amount of formazan produced is directly proportional to the number of living cells. The absorbance of formazan was measured at 450 nm and enables the calculation of viable cells percentage for each treatment compared to non-treated cells ( Table 1) . The technique of quantibody assay is similar to the method described in our previous studies. 1,11 During the incubation time, the volume of medium varied differently in each well and the volume collected might be reduced. In order to normalize the profiling results, cell culture medium was added to the different collected media to reach 200μl before performing the profiling assay. Then, the medium samples were tested undiluted on arrays. The results of cytotoxicity assay showed there was no strong effect of treatment on the viability of the T cells even though the viability went down until 87% for cells treated 72h with 0.01% rose extract. Indeed, such a decrease of viability in one tested replicate is not statistically representative of a cytotoxicity effect of the rose extract. Figure 1 shows the results of cytotoxicity assay. IFN-gamma, MCP-1, GRO, RANTES and TIMP, Angiopoietin 1 and MMP-9 were elevated. Except MMP-9 which had fold changes >2, other cytokines were mildly elevated at various concentrations and timing of rose extract treatment (Table 2 and The results showed that the cytokines GRO, IFNg, IL-6, MCP-1, RANTES, TGF-b1, TIMP-1, TIMP-2, Angiopoietin-1, Angiopoietin-2, G-CSF, IL-10, IL-1a, MMP-9 and VEGF R2 were enough detected to enable the calculation of one-fold change at least. In this study, the Angiopoietin 2 27 and VEGF R2 28 are involved in angiogenesis and inflammation. G-CSF has a role in granulopoiesis and anti-inflammation. 29 TIMP-2 30,31 and TGF-b are mildly elevated. 32 TIMP2 actively attenuates the negative remodelling and TGFb2 is involved in tissue repair. 33 The catalytic effects of the rose extract on the immune system also needs to be studied, and since the performance invitro is unique it could act as a 'salt' in the immune functions, though speculative in the current scenario. This study was performed invitro with limited number of samples and numbers. Further extensive evaluation of the extract needs to be performed in large numbers invitro and later in animal models. The side-effect profile of the extract also needs to be evaluated. Also, the feedback mechanism, which could exist in these T-cells in immune regulation need to be studied in vivo. 34 . The quality of the rose extract needs to be standardised by HPLC etc. In this study, the effect of red rose extract on the secretion of cytokines from T cells CD4+ was assessed. The secreted cytokines were profiled after 48h or 72h of incubation. The results showed that the cytokines GRO, IFNg, IL-6, MCP-1, RANTES, TGF-b1, TIMP-1, TIMP-2, Angiopoietin-1, Angiopoietin-2, G-CSF, IL-10, IL-1a, MMP-9 and VEGF R2 were enough detected to enable the calculation of one-fold change at least. There was no significant fold change over 2 or lower 0.5 except MMP-9 with fold change often over 2. 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