## 3 Results
### Transcriptomic Analysis (RNA-Seq)  
Using next-generation sequencing, RNA-seq is a powerful tool capable of detecting the quantitative measurement of RNA abundance or gene expression [24]. RNA-seq analysis of the hippocampus tissue was used to compare the differential gene expression pattern of the vehicle group compared to SFPWH-treated and fluoxetine-treated groups. As shown in Figure 4a, 72 detected subclusters and 82 transcripts. Gene ontology enrichment analysis (Figure 4b) was performed by ShinyGO v0.741 [25], revealing the top pathways for the detected genes. Data analysis showed a significant differential gene expression pattern in 22 genes, which might be potentially related to mood disorders (Figure 4c).  
The expression pattern of the mean SFPWH (50 mg/kg) group replicates revealed a significantly different gene expression compared to the control group. Where 11 transcripts were downregulated in genes (Ube2j2, Dctn6, Nor1, Acp2, Tuba1c, Gm14150, Actr3-ps, Grik1, Commd5, and Gm3226). In addition, 7 transcripts were upregulated in genes (Msmo1, Casp9, Hspel-rs1, Tmem44, Rps13-ps2, Rps3a2,and Dctn6). In comparison to the control group. At the same time, fluoxetine-treated group showed a significantly different gene expression in six genes where two transcripts were downregulated in genes Cacan1h and Dynci12. Moreover, five transcripts were upregulated in genes (Rbl2, Mas1, Hyi, Morf4l2, and Dynci12). SFPWH (150 mg/kg) group has not shown any significant gene expression compared to the control group (Table 2).  
Figure 3: Effect of safflower water extract treatment on spontaneous alternation in YMT. Each mouse was allowed to explore the Y maze in an 8 min session. The number of alternations between 3 arms was calculated and reported as a percentage of the total number of entries. Each bar represents the mean \(\pm\) SEM (n = 8). * and ** indicates significantly different at \(p<\) 0.05 versus untreated group, Student \(t\)-test.  
Figure 4: _Cont._  
Figure 4: Transcriptome differences detected between the mean of the control group and treatment groups. (**a**). Hierarchical cluster analysis of gene expression based on log-ratio FPKM data for the mean of transcripts replicates illustrating the impact of the administration of safflower hot water extract and fluoxetine treatment in comparison to control where red indicates upregulation pattern while blue color indicates downregulation pattern. (**b**). Heatmap showing significantly different gene expression patterns (log2 fold). (**c**). The bar charts for the gene ontology enrichment analysis show the top pathways for the significant enrichment genes for all groups. \(p\)-value cutoff (FDR) 0.05.  
\begin{table}
\begin{tabular}{l c c c c c} \hline \hline
**Gene** & \multirow{2}{*}{**NCBI ID**} & \multirow{2}{*}{**log2 Fold**} & **Gene** & \multirow{2}{*}{**NCBI ID**} & **log2 Fold** \\
**Symbol** & & & & **Symbol** & \\  & **SFPWH 50 (mg/kg)** & & & **Fluoxetine (10 mg/kg) Groups** & \\ \hline _Ulbe2j2_ & 140,499 & \(-\)2.06 & _Cacan1h_ & 58,226 & \(-\)1.42 \\ _Dcnt6-206_ & 22,428 & \(-\)1.90 & _Dync12-203_ & 13,427 & \(-\)0.97 \\ _Ncor1_ & 20,185 & \(-\)1.43 & _Rb12_ & 19,651 & 1.37 \\ _Acp2_ & 11,432 & \(-\)2.42 & _Mas1_ & 17,171 & 1.24 \\ _Tlua1c_ & 22,146 & \(-\)1.87 & _Dync112-205_ & 13,427 & 1.22 \\ _Gm14150_ & 100,043,840 & \(-\)1.56 & _Hyi_ & 68,180 & 1.47 \\ _Actr3-ps_ & 667,799 & \(-\)1.46 & _Morf412_ & 56,397 & 2.23 \\ _Grik1_ & 14,805 & \(-\)1.29 & & & \\ _Commd5_ & 66,398 & \(-\)2.68 & & & \\ _Gm3226_ & 100,041,240 & \(-\)2.14 & & & \\ _Msm01_ & 66,234 & 0.32 & & & \\ _Casp9_ & 12,371 & 2.67 & & & \\ _Hye1-rs1_ & 628,438 & 2.35 & & & \\ _Tmem44_ & 224,090 & 1.51 & & & \\ _Rps13-ps2_ & 100,039,924 & 2.83 & & & \\ _Rps3a2_ & 100,043,780 & 4.56 & & & \\ _Dcnt6-202_ & 22,428 & 1.84 & & & \\ \hline \hline \end{tabular}
\end{table}
Table 2: List of the significant differential expressed genes of safflower hot water extract SFPWH (50 mg/kg) and fluoxetine-treated group compared to the control group.