*2.3. Ech A A*ff*ects the Expression of Cell Survival-Related Molecules of Rat Ischemic Stroke Brain*

To demonstrate the mechanism of physiological improvements, which include the restoration of the damaged brain region and the intensification of attenuated behavior after Ech A treatment, we focused on the occurrences after ischemia reperfusion injury. Ischemia reperfusion-injured brain suffers from oxidative stress and induces the cell death regulating pathway [19]. Considering this, we investigated the expression levels of cell viability-related factors in our experimental animal model, including Bcl-2, Caspase-3, Bax (Figure 3A), p-ERK/ERK, p-AKT/AKT (Figure 3B), and brain-derived neurotrophic factor (BDNF) (Figure 3C). The effect of Ech A treatment on the expression of these regulators was estimated in the brain tissue of MCAo rats. Bcl-2, caspase-3, and Bax work as major mediators for cell survival and death and are activated by various stimuli [18,19]. Bcl-2, an apoptosis inhibitor, is a key player in the mechanism of anti-apoptosis [20,21]. In contrast, caspase-3 and Bax are pro-apoptosis molecules which signify the onset of apoptosis [21]. Compared to the control group, Ech A treatment in the MCAo rat model significantly increased the expression level of Bcl-2 and decreases the levels of caspase-3 and Bax. The extracellular signal-related kinases (ERK) are essential regulators associated with vital cellular functions, including cell proliferation, differentiation, migration, senescence, and apoptosis in the generic mitogen-activated protein kinase (MAPK) signaling pathway [22]. Furthermore, in the PI3K/AKT/mTOR signaling pathway, AKT is also a core component of various processes of cellular activities, including nutrient uptake, anabolic reactions, metabolism, cell growth, proliferation, differentiation, apoptosis, and survival [23]. Our results indicate an increase in the expression levels of p-ERK/ERK and p-AKT/AKT in the Ech A-treated MCAo rat model as compared to the control group. The brain-derived neurotrophic factor (BDNF) significantly supports

neuronal differentiation and survival, synaptic formation and plasticity, and neurogenesis, and has been widely researched in various neurological conditions [24]. Our studies reveal increased BDNF expression in the brain of MCAo rat model after exposure to Ech A, as compared to the control group. Taken together, these findings confirm that Ech A relieves the physiological decline in the MCAo rat model by increasing and supporting cell survival in the injured brain region.

**Figure 3.** Ech A treatment in cerebral injured brain alters the expression levels of cell viability-related factors. (**A**) The protein expression levels of cell survival and death regulators, such as Bcl-2, caspase-3, and Bax. (**B**) The protein expression levels of key players in vital cellular function regulation pathways, such as ERK and AKT. (**C**) The mRNA expression level of BDNF, supporting cell survival alteration in the injured brain region (\* *p* < 0.05).
