Molecular Mechanism Study of Vitorgan Cell Molecular Therapy

VitOrgan is a cell molecule drug extracted from bovine embryonic cells for anti-aging therapy since the 1965s in European countries. However, the molecular mechanisms involved remain unclear and underresearched. In this study, we applied human cytokine arraywith a group of 440 against-cytokine antibody, functionalGO term enrichmentand network technique to analyze the composition of vitOrgan(Mixed liquid: NeyPson Nr.5, NeyThel Nr.62, NeyNormin Nr.65, NeyDIL Nr.66, NeyDia Nr.67, NeyDIL Nr.70, NeyDesib Nr.78, NeyTroph Nr.96). The experimental results confirmed that the vitOrgan drug contains 123 cytokines, including ACE2, ADAM8, ADAM9, ADAMTS13, AFGF, ANGPT1, ANGPT2, ANG4, AR,AXL, BAFF,2B4,TNFRSF17, BDNF, bFGF, BMP4, BMP5, BMPR1A,BMPR1B, BMPR2,CDH4,CDH13, P-Cadherin, CD40L, CD84, CD229, CTLA4, CXCL9, CXCL10, CTA, TNFRSF6B, DCN, ENA-78,FGF19, FGF21, FOLR1, FSH, GPRASP1, GPRASP2, GCP-2, GITRL,HCC4,ICAM3,IFNA1,IFNB1,IFNG,IGF1,IGF2,IGFBP3,IGFBP4,IGFBP5,IGFBP6,IL1A, IL2, IL3, IL4, IL-10, IL-13, IL-15, IL-17A, IL-18, IL-21, IL-27, IL36RN, IL1RI, IL1RAP,IL1R4, IL10RB, IL13RA2, IL17RA, IL17BR, IL-17C, IL21R, F11R, JAM2, KLK14,LAG3, LDLR, LEP, LIF, CCL2, CCL8, CCL7, CL22, MER, MICA, MICB, CXCL9, CCL4, MMP-2, MMP-3, NACM1,MME,NOTCH1,NTF4,PDCD1, PECAM-1, PGYRP1,TGFB3,TG,TEK,HAVCR2, TLR4, TNFAIP3, TWEAK, VEGF, VCAM1, WISP-1, EDA2R and ULBP2.

Rothschild also applies vitOrgan in the field of beauty and cosmetics, resulting in the birth of biolifting anti-aging therapy (https://vitorgan.de/). Neurodegenerative diseases are devastating and affect an estimated 1 billion individuals worldwide [9]. It is anticipated that cell-based drugs may alleviateor even reverse the progression ofneurological diseases [9]. In 2018 bovine myoblast cell productionin a microcarriers-based system was reported to provide valuable insights forclinical relevant cell therapy [10].
More than 40 years of clinical experience of vitOrgan has been accumulated targeting skin cosmetology, anti-aging, organ function maintenance, disease risk intervention, chronic disease adjuvant therapy and others. Cytokines are glycoproteins that help coordinate many physiological functions, including immune function, inflammation, hematopoiesis, homeostasis, and tissue repair [11,12].
Cytokine classification mainly include interferon (IFN-γ), interleukin (IL), colony stimulating factor (G-CSF) and erythropoietin (EPO) [11,13]. From 1982, the invention and productionof recombinant insulinto 1997 G-CSF became the first peptidedrug with annual sales exceeding 10 billion US dollars. Clinical indications of cytokines as supplementary therapy has relatively positive effects [14,15]. VitOrgan as live or fresh cell therapy is not only used in adjuvant treatment of daily diseases such as asthma, bronchitis, fatigue syndrome, sinusitis, immunodeficiency, diabetes mellitus, cardiovascular and cerebrovascular diseases, but also in the prevention and treatment of chronic motor diseases such as osteoarthritis, cartilage injury, intervertebral disc disease, etc. It is widely used in the field of anti-aging, preventative medication to reduce the risk of diseases. After treatment, the symptoms of many patients were significantly alleviated or even disappeared.
Manypeople experience the improvement in the energy level, physical and sexual function, and organ function, which may indicate vitOrgan's potential to delay the aging process [data under press]. Combining with the positive clinical outcomes from Germany, Brazil, Thailand, the Philippines, Austria, the United States, Russia, Colombia and othercountries, it is important to share the encouraging research data of vitOrgan with the research community. However, the molecular fraction and mechanism of vitOrgan is still unknown. In this study, we used cytokine microarray to detect the components of vitOrgan. Clustering and interaction analysis of vitOrgan-related cytokines were carried out using biomedical informatics technology through software in Database for Annotation, Visualization and Integrated Discovery (DAVID). These experimental data further providedmolecular basis for elucidating the mechanism of vitOrgan-related factorstherapy.
Thus, our data would provide evidencefor cell and molecule therapy targetingtissue and organ to protect aging and damage.

Materials
The concentrated injection solution, vitOrgan of NeyPson Nr. 5

Gene Ontology and Pathway Analysis
Gene ontology (GO) and pathway analysis are suitable methods for integration genes with biological interaction and pathway networks to detect coordinated changes in functionally related genes. All the target genes are subjected to GO and pathway analysis in order to describe functional association of target genes.
GO analysis was performed using GO pathway analysis using the open access software DAVID bioinformatics system and database (Database for Annotation, Visualization and Integrated Discovery, http://www.david.abcc.ncifcrf.gov website).Design and realization of software of cytokine statistical analysis for evaluation based on R language through KEGG (Kyoto Encyclopedia of Genes and Genomes https://www.genome.jp/kegg/) [16,17].

Data Analysis
Protein similarity analysis used the database of UniProt (https://www.uniprot.org/).A preliminary approach to the whole vitOrgan cytokines consisted of an elaboration for Network analysis with IPA software. As proteins in biological systems seldom work as independent entities, rather they are placed in widely interacting regulatory networks, the study of protein-protein interactions is a valid instrument to exploit in silico modelling to retrieve biologically relevant insights, through the individuation ofpivotal webs of proteins and their potential main modulators. Network analysis software, such as IPA, determines and graphs unbiased networks, in which gene products are represented as nodes, and the biological relationship between two nodes is represented as an edge (line). All edges are supported by at least a reference from the literature, from a textbook, or from canonical information stored in the Ingenuity Pathways Knowledge Base for IPA [16,17]. The databases and website including STRING (https://string-db.org/), National Center for Biotechnology Information (https://www.ncbi. nlm.nih.gov/) and GeneCardsSuite (https://www.genecards.org/).

Statistical Analyses
Data extraction can be done using the GAL file that is specific for this arrayalong with the microarray analysis software (GenePix, ScanArray Express,ArrayVision, MicroVigene, etc.).Comparison of means between two groups was accomplished by the Student's t-test (two tailed). A P value <0.05 was considered statistically significant.

Cytokine Expression Profiles
To identify the cytokines contained in vitOrgan injection  (Table 1).

GO Annotation Analysis of vitOrgan Cytokines
Network analysis through DAVID yielded the identification of 91 networks, as reported in Table 2 Table 2.

Networks on the Whole Interactome of vitOrgan Cytokines
The networks of DAVID analysis on whole vitOrgan cytokines are shown in Figure 2.  Gene ontology (GO) and KEGG pathway classification of target genes. The x-axis shows the GO categories and y-axis shows the target genes. The x-axis shows the P-value and y-axis shows the diverse biological functions of target genes according to the GO categories. This interaction showed 68 cytokines of vitOrgan and the red box labeled molecular denoted up-regulated cytokines, relating with chemokines (CCL25, CCL4,CCL4L1, CCL4L2, CCL17, CCL5,   CCL8, CCL16, CCL7, CCL2, CCL12), CXC subfamily (CXCL5, CXCL6,   CXCL9, CXCL10, CXCL12), the class I helical cytokines (IL-2, IL-4,   IL15, IL3, IL4, IL13,IL27A, LIF), the class II helical cytokines(IL10,   IL22  (iv) Reproductive tract (Testican 2) ( Figure 1) and ( Table   1) [16]. Nevertheless, most cytokines of vitOrganhave been described acting on the whole body (Table 2) (ii) Lipid metabolism, molecular transport, small molecule biochemistry (TGFB1, TGFB2, TGFBI, TGFBR3, THBS1, LEP,   BTC, EGFR, TGF-β2, IGFBP3, IGFBP1, IGFBP2, IGFBP4, IGFBP5 Six pathways that shared ten cytokines between vitOrgan and bovine milk were listed above (i-vi) while the others with less shared cytokines were not listed. Because of such relevance, vitOrgan may help repair the damage to tissues and organs resulting from aging and environmental hazard, similar to the molecular mechanisms of bovine milk [16]. It would be helpful to study natural medicine in a scientific and systematic fashion to unveil the underlying mechanisms, which would point in the right development direction of natural medicine and complementary and alternative medicine in the future.
Furthermore, the results of such a large biomedical data analysis require further clinical evaluation.
As we known live cell therapy(LCT)is an alternative treatment without medical evidence of effectiveness, which is through online marketing worldwide. It was reported the intramuscular injections of cell suspensions from fetal sheep,including young rams and pregnant ewes, were injected to human recipients for rejuvenation (anti-aging) and other ailments with positive feedback [20]. Apart from Germany recipients, medical tourists from North America and Asia travel to Germany to receive injections. Although several incidents were reported that LCT was the possible cause for the Q fever in Canada, Germany and the United States, vitOrgan have not experienced the issue. When working with vitOrgan, the health practitioners worldwide should pay more attention to the potential risk factors for LCT induced Q fever [21,22].
Altogether, this study focused on the cytokines involved in vitOrgan and is by far the first investigation about the molecular composition of vitOrgan injection solution. We have found 123 cytokines using human array and 91 important signal pathways associated with development, proliferation, differentiation, immunology and metabolism. Moreover, a preliminary map of vitOrgan proteins interactome was also constructed, which can greatly help understand the mechanism of vitOrgan. With more indepth knowledge of vitOrgan, clinician can put it into more targeted clinical applications and popularize the use of vitOrgan in the world.