# Comparative Study on the Role of Berberine and _Berberis_
## 3 Results and Discussion
### Immunohistochemistryistochemistry Analysis of CD1 Expression  
Previously, [PERSON] et al. indicated negative regulation of the PI3K/AKT pathway by berberine. Despite much research, the precise mechanism of action of berberine on molecular and cellular targets remained unknown [38; 39]. We sought to find out the role of berberine in the protein expression of cyclin D1 in liver tissue specimens. Genetic mutations in cell cycle regulatory machinery that direct transit over the G1 phase occur frequently in many types of human cancers, and one of the most common alterations is the overexpression of cyclin D1 [40]. CD1 activates its cognate CDK (4/6). Moreover, it is also an important target of the STAT signaling pathway in many types of cells, and it regulates metabolism and fat cell formation. Thus, it provides an insight into the role of metabolism in human cancer [30]. Previously, our in vitro experiments have shown that _B. lycium_ Royle ethanol-based extract suppressed the expression of CDK1 genes, along with downregulating other cyclin-dependent kinases [11]. The cell cycle is regulated by cyclin-dependent kinases that are activated by cyclins. The development of cells and cell division is regulated by cyclins and cyclin-dependent kinases [41]. The overexpression of these positive regulators (cyclins) of the cell cycle enhances the process of tumorigenesis in many types of cancer [31], and may lead to HCC progression from cirrhosis [42]. Thus, we wanted to investigate whether berberine and BLE have any regulatory roles associated with CD1 markers in vivo. As they can impart a crucial role in growth, proliferation and metastasis of the tumor in other tumor types [31; 38; 39], they can, therefore, be important diagnostic/therapeutic markers in HCC patients. In the current study, we found that BLE treatment at low (60 mg/kg) and high (120 mg/kg) doses negatively regulated the expression of CD1, and berberine also downregulated this expression well (IHC score of 3.3), even more significantly at a higher dose (120 mg/kg). This proves that berberine at 120 mg/kg of mice b.wt significantly suppressed cyclin D1 expression (Figure 2). Therefore, it decreased the activity of CDK and halted the proliferation of cancer cells, resulting in suppression of HCC development from the liver injury. Berberine (C\({}_{20}\)H\({}_{19}\)NO\({}_{5}\)) is a multitarget drug that acts via several mechanisms, thus inducing apoptosis and autophagy, preventing metastasis and angiogenesis in various cancer cells, including liver cancer cells. The pro-apoptotic effect of berberine is generated by extrinsic and intrinsic pathways. Berberine can increase ROS production in cells, which activates caspase 12 and activates BCL-2 proteins. It acts via Fas, MAPK and AMPK pathways, and ultimately causes apoptosis of liver cancer cells [41]. The comparison of the HCC control group and the treatment group, on the basis of immunohistochemistry scores, is given in Table 3. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**No. of Days of DEN** & **Behavior/Symptoms of** & **Weight of Dead** & **No. of Mice Died** \\
**Treatment** & **Dead Mice** & **Mice (g)** & **No. of Mice Died** \\ \hline
0 & Normal & 18–22 & 0 \\
30 & Loss of appetite & 34–42 & 0 \\
35 & Sudden weight loss & 28–33 & 1 \\
40 & Systemic inflammation & 27–32 & 2 \\
45 & Red eyes & 26–28 & 4 \\
50 & Fast breathing and shivering & 24–30 & 5 \\
60 & Died & 20–24 & 8 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Animal burden and weight loss upon application of DEN and CCl\({}_{4}\) in mice, who started dying on day 35 of carcinogenic chemical application. Figure 2: Immunohistochemistry analysis of cyclin D1 (CD1) expression in liver tumor tissue sections.