*2.8. Effect of OPs on The Expression of Hepatic COX-2, MIP-2, NF-κB, and p-ERK in Cd-Exposed Mice*

COX-2 is a key enzyme in initiating hepatic inflammatory reactions [28]. Meanwhile, macrophage inflammatory protein (MIP)-2 is a potent neutrophil attractant and activator, contributing to the pathogenesis of inflammatory diseases [29]. MIP-2 and COX-2 would be elevated in Cd-induced inflammation [30]. As shown in Figure 6A, enhanced COX-2 and MIP-2 staining were observed around the central vein of hepatocytes in the Cd-exposed group. OPs treatment noticeably reduced the hepatic COX-2 and MIP-2 levels.

In the process of Cd-induced inflammation, the extracellular signal-regulated kinase (ERK) signal pathway would be activated, and the nuclear factor-κB (NF-κB) subsequently was up-regulated [31]. Western blotting assays illustrated that the expression of NF-κB and p-ERK were highly induced in the Cd-exposed group. However, the OPs treatment effectively dampened the expression of NF-κB and p-ERK (*p* < 0.01; Figure 6B,C). The above results implied that OPs might alleviate hepatic inflammation by inhibiting the expression of inflammatory activators (COX-2 and MIP-2) and related inflammatory pathways (NF-κB and ERK).

**Figure 6.** Effect of OPs on the expression of COX-2, MIP-2, NF-κB, and p-ERK in the liver. (**A**) The expression of COX-2 and MIP-2 in the liver by Immunohistochemical (IHC) Staining; (**B**) Western blot analysis of NF-κB and p-ERK proteins, Cd was sampled in 10 μL and 20 μL volumes, respectively; (**C**) Quantitative densitometric analysis of NF-κB and p-ERK proteins. These Data are expressed as the mean ± SEM, *n* = 6 in each group. Compared with the control group, \*\* *p* < 0.01; compared with the Cd group, ## *p* < 0.01.

## *2.9. Effect of OPs on Hepatic Apoptosis in Cd-Exposed Mice*

Apoptosis-related mitochondrial Bcl2-associated X protein (Bax) and Caspase-3 are two important pro-apoptotic factors. Under the stimulation of oxidative stress caused by Cd, the hepatic Bax increased, then the downstream Caspase-3 was up-regulated, and eventually, apoptosis occurred [32]. Anti-apoptotic Bcl-2 plays a central regulatory role in apoptosis [33]. Accordingly, we examined the effect of OPs on Cd-induced hepatic apoptosis by measuring the levels of pro-apoptotic factors (Bax and caspase-3) and antiapoptotic factor Bcl-2 in Cd-exposed mice. As shown in Figure 7A–C, Cd significantly decreased the levels of anti-apoptotic Bcl-2 but increased the levels of pro-apoptotic factors (Bax and caspase-3) (*p* < 0.01). On the contrary, OPs treatment significantly increased Bcl-2 while decreasing Bax and caspase-3 levels in Cd-exposed mice (*p* < 0.01). The qRT-PCR results showed that OPs significantly induced the expression of Bcl-2 while suppressing the expression of Bax and caspase-3 in Cd-exposed mice (*p* < 0.01). OPs exhibited a strong anti-apoptotic effect on Cd-induced apoptosis in mice.

Cd can regulate the PI3K/AKT signaling pathway to induce apoptosis [34,35]. Additionally, PI3K/AKT signaling pathway also plays a crucial role in the regulation of inflammatory protein expressions (COX-2 and MIP-2) [36]. Western blotting assays demonstrated that Cd exposure led to the elevation of the expression of PI3K and p-AKT, accompanied by the imbalance of pro-/anti-apoptotic proteins (Bax, caspase-3 and Bcl-2) (*p* < 0.05). By contrast, the OPs treatment inhibited the activation of the PI3K/AKT signaling pathway and restored the balance of pro-/anti-apoptotic proteins in Cd-exposed mice. The results implied that OPs might alleviate hepatic apoptosis by restoring the balance of pro-

/anti-apoptotic proteins via inhibiting the PI3K/AKT signaling pathway in Cd-exposed mice.

**Figure 7.** Effect of OPs on apoptotic marker levels and mRNA expression of Cd-induced mice in the liver. (**A**) Bax concentration; (**B**) Caspase-3 activity; (**C**) Bcl-2 concentration; (**D**) Relative mRNA expression levels of Bax, caspase-3 and Bcl-2; (**E**) Western blot analysis of PI3k, p-AKT, Bax, caspase-3 and Bcl-2 protein expression, Cd was sampled in 10 μL and 20 μL volumes, respectively; (**F**) The quantitative densitometric analysis of PI3k, p-AKT, Bax, caspase-3 and Bcl-2. These Data are expressed as the mean ± SEM, *n* = 6 in each group. Compared with the control group, \* *p* < 0.05, \*\* *p* < 0.01; compared with the Cd group, # *p* < 0.05, ## *p* < 0.01.
