*2.3. SR, CR, and Combined Extracts Alleviated Inflammation by Regulation of Pro-Inflammatory Cytokine Expressions through MAPK Signaling Pathway*

Recently, many studies have shown that pro-inflammatory macrophage accumulation in liver could trigger chronic low-grade inflammation that promoted the development of insulin resistance [21]. On day 30 after STZ injection, levels of CRP, IFN-γ, TNF-α, IL-6, resistin, SOCS3, IL-1β, and NO in serum were significantly increased in the model group compared to those in the normal group. However, the levels of these pro-inflammatory cytokines were reduced to a range from 96.0% to 58.4% after oral administration of metformin, SR, CR, LSC or HSC (Figure 3). MAPK pathway plays an important role in the inflammatory process [22], so the key targets in this pathway were investigated in this study. As the qPCR results show in Figure 4A–G, mRNA expressions of P38, ERK, c-jun, c-fos, JNK, IKK and P65 were significantly increased in the model group. With the treatment of metformin, SR, CR, LSC, and HSC, mRNA expressions of the above major targets in MAPK pathway were remarkably decreased to a range from 87.9% to 68.8%. Besides, the effects of LSC and HSC were more significant than SR and CR, while metformin showed a similar effect to HSC. NF-κB/p65 is a key transcriptional factor of inflammation regulated gene expression of pro-inflammatory cytokines. Further studies indicated that P65 was activated by phosphorylation in the model group, which could be suppressed by metformin, SR, CR, LSC, and HSC (Figure 4H).

**Figure 3.** Serum contents of C-reaction protein (CRP), interferon gamma (IFN-γ), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), resistin, nitric oxide (NO) and suppressor of cytokine signaling 3 (SOCS3) in the normal group (N), model group (M), and groups gavaged with metformin (P), scutellaria Radix (SR), coptidis Rhizome (CR), low dose of combined extracts group (LSC) and high dose of combined extracts (HSC). The values were shown as mean ± SD. # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. normal group; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. model group. Data were analyzed by One-way-ANOVA.

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**Figure 4.** The effect of metformin (P), scutellaria Radix (SR), coptidis Rhizome (CR), low dose of combined extracts group (LSC) and high dose of combined extracts (HSC) on MAPK signaling pathway. (**A**–**G**) mRNA expression levels of c-jun N-terminal kinase (JNK), p38 mitogen-activated protein kinases (P38), extracellular regulated protein kinases (ERK), c-fos, c-jun, Inhibitor of nuclear factor kappa-B kinase (IKK) and P65 in liver of the N, M, P, SR, CR, LSC, and HSC by qPCR; (**H**) Protein levels of P65, phosphorylated P65 (p-P65) in liver of the N, M, P, SR, CR, LSC, and HSC by Western blot. The values are shown as mean ± SD. # *p* < 0.05, ## *p* < 0.01, ### *p* < 0.001 vs. normal group; \* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001 vs. model group. Data were analyzed by One-way-ANOVA.
