## 4 Discussion  
_P. grandiflora_ has great potential for medicinal use in various diseases due to different pharmacological properties. Immune enhancement is one of the major outcomes of PGE treatment, which is considered to be one of the important factors behind the positive conclusion in different cancers and other diseases such as cough, phlegm and tuberculosis [1]. The immune-enhancement mechanism of PGE is encouraging the researchers to elucidate the PGE and/or its major constituents (platycodin D, etc.) as a possible treatment in many chronic diseases [1]. As immune enhancement is a complex process, its mechanism is currently attributed to the combined effect of several genes and different pathways. Different studies have reported immune enhancement in both innate and adaptive immune responses through increased activity of immune-system cells and molecules [5; 6; 7]; no study has been carried out to study immune enhancement through whole transcriptome analysis in PGE-treated organisms, which can elucidate the molecular mechanism and genes behind the immune enhancement. Hence, to study the role of PGE on the whole transcriptome for immune enhancement, we analyzed the PGE-treated RNA-Seq datasets from PGE-treated mice in two comparisons. In comparison-II, the mice fed on PGE and HFD were compared with mice fed on HFD as well as normal diet. Having both normal and high-fat diet mice in PNTG group in comparison-II made this comparison more challenging, but it was necessary to minimize the effect of HFD in PGE-treated mice. The possibility of interaction between the HFD and PGE treatment cannot be ruled out, but unavailability of transcriptome data from normal PGE-treated organisms limits the current study. Thus, this preliminary study must be further extended according to availability of datasets in the future. The animal study with normal diet and PGE treatment can be used to generate whole transcriptome datasets that would be most appropriate for the immune-enhancement study. Furthermore, we included  
Figure 6: Protein–protein interaction network between 126 down-regulated immune-suppressor genes. all of the nine samples in comparison-II, which increased the statistical significance of this study. High alignment rate was achieved in the alignment of reads with the reference genome (>98% overall alignment rate) in all nine samples, which signposted the good quality of samples used in the study (Table 1). The state-of-art RNA-seq analysis tools such as HISAT2, StringTie, iDEP9.1, DESeq2 and EdgeR were used [21; 23] for different assembly and expressions analysis (Figure 1). Almost all (127 out of 130) up- and (853 out of 859) down-regulated genes in comparison-II were common with up- and down-regulated genes in comparison-I, which suggest the common pattern in both comparisons (Table D1 and Table D2). Siglec-15 was one of most down-regulated genes in both comparisons and was recently discovered as the therapeutic target in normalization cancer immunotherapy [34]. Down-regulation of Siglec-15 may be the main reason behind both immune enhancement and anticancer activity of PE. In a very recent landmark study, down-regulation of this immune-suppressor gene was found to suppress antigen-specific T cell responses in both in vitro and in vivo conditions [34]. Blockage of Siglec-15 through specific monoclonal antibodies reversed the suppression of T cells, enhanced tumor immunity and inhibited the growth of tumors in multiple established tumor models [34]. Down-regulation of Siglec-15 protein molecules can also be responsible for immune-enhancement activity of other plant extracts or compounds of unknown mechanism. Further research is highly required to study this candidate gene/protein. All DEGs were used for the functional enrichment analysis through prediction of five important biological categories to predict genes important in immunity-related functions.