**2. Results**

*2.1. PE*/*HDP Patient Sera Up-Regulated Gene Expression of IL-6, CCL2, High Mobility Group Box (HMGB)1, S100 Ca2*+*-Binding Protein B (S100B), and Receptor for Advanced Glycation Endproducts (RAGE) in Primary Cultured Human Adipocytes*

Obesity increases PE/HDP risk. Maternal obesity, increased insulin resistance, and inflammation are involved in the pathogenesis of PE/HDP [24,25]. Furthermore, PE/HDP risk has been reported to increase 2–4-fold among women with diabetes [26]. We therefore hypothesized that the PE/HDP patient sera contain some of these factors that induce insulin resistance and/or inflammation. We incubated primary cultured human adipocytes with sera from disease-free pregnan<sup>t</sup> women (control) or those from PE/HDP (patients) for 24 h, and the gene expression of *IL-6*, *CCL2*, *tumor necrosis factor* α (*TNF*α), *leptin* (*LEP*), *adiponectin* (*ADIP*), *resistin* (*RETN*), *HMGB1*, *S100B*, and *RAGE* in the adipocytes was measured via real-time reverse transcriptase-polymerase chain reaction (RT-PCR). As shown in Figure 1, mRNA levels of *IL-6*, *CCL2*, *HMGB1*, *S100B*, and *RAGE*, but not *TNF*<sup>α</sup>, *LEP*, *ADIP*, and *RETN* (*P* = 0.4496, *P* = 0.1157, *P* = 0.0875, and *P* = 0.2912, respectively) were significantly increased by the addition of PE/HDP patient sera compared to those cells incubated with control sera.

#### *2.2. Up-Regulation of IL-6 and CCL2 by HMGB1 and Advanced Glycation Endproducts (AGE) in Adipocytes*

It is well-known that HMGB1 and S100B are typical ligands for RAGE. RAGE expression was reported in adipocytes and SW872 cells [27,28], and furthermore immunofluorescent staining of RAGE in 3T3-L1 adipocytes was shown [27]. RAGE expression was up-regulated by ligands for RAGE [29], we tested whether ligands for RAGE up-regulate gene expression of inflammatory mediators, such as *IL-6* and *CCL2*, in human SW872 adipocytes. We added HMGB1, AGE, and S100B in SW872 culture medium, incubated for 24 h, and the expression of *IL-6* and *CCL2* was analyzed via real-time RT-PCR. As shown in Figure 2, mRNAs of *IL-6* and *CCL2* were significantly up-regulated by the addition of HMGB1 and AGE. In contrast, S100B, another noted ligand for RAGE, failed to up-regulate mRNA for *IL-6* or *CCL2*.

In order to see whether the up-regulation of mRNAs for *IL-6* and *Ccl2* occurred only in SW872 or other adipocytes, we cultured mouse 3T3-L1 preadipocytes, di fferentiated them into di fferentiated adipocytes, and tested whether ligands for RAGE up-regulate gene expression of *IL-6* and *Ccl2* in mouse 3T3-L1 undi fferentiated preadipocytes and di fferentiated adipocytes. As shown in Figure 3, the mRNA levels of *IL-6* were significantly up-regulated by AGE and HMGB1 but not by S100B (*P* = 0.6414) in di fferentiated 3T3-L1 adipocytes, but unchanged by any of the RAGE ligands (AGE, HMGB1, or S100B) in undi fferentiated preadipocytes ( *P* = 0.8037 [No addition vs. AGE], *P* = 0.4793 [No addition vs. HMGB1], and *P* = 0.3138 [No addition vs. S100B]). In contrast, the mRNA levels of *Ccl2* remained unchanged in response to AGE, HMGB1, or S100B in 3T3-L1 di fferentiated adipocytes

(*P* = 0.1892 [No addition vs. AGE], *P* = 0.2885 [No addition vs. HMGB1], and *P* = 0.4024 [No addition vs. S100B]), but significantly up-regulated in the undifferentiated preadipocytes by the addition of AGE but not by HMGB1 and S100B (*P* = 0.1241 [No addition vs. HMGB1] and *P* = 0.4305 [No addition vs. S100B]) (Figure 3). Previous studies reported that S100B up-regulated TNFα in adipocytes [30,31]. In contrast, S100B induced neither *IL-6* nor *CCL2* in adipocytes in this study, suggesting that SW872 and 3T3-L1 cells may insensitive to S100B.

**Figure 1.** The mRNA levels of *IL-6*, *CCL2*, *TNF*<sup>α</sup>, *LEP*, *ADIP*, *RETN*, *HMGB1*, *S100B*, and *RAGE* in primary cultured human adipocytes treated with sera from disease-free control (Control) or preeclampsia/hypertensive disorders of pregnancy (PE/HDP) patients (Patients) for 24 h. The levels of the mRNAs were measured via real-time reverse transcriptase-polymerase chain reaction (RT-PCR) using β*-actin* as an endogenous control. Data are expressed as mean ± SE for each group (*n* = 4). The statistical analyses were performed using Student's *t*-test.

**Figure 2.** The mRNA levels of *IL-6* and *CCL2* in SW872 human adipocytes treated with 1 μg/mL HMGB1, 150 μg/mL advanced glycation endproducts (AGE), or 100 ng/mL S100B for 24 h. The levels of the mRNAs were measured via real-time RT-PCR using β*-actin* as an endogenous control. Data are expressed as mean ± SE for each group (*n* = 4). The statistical analyses were performed using Student's *t*-test vs. No addition.

**Figure 3.** The mRNA levels of *IL-6* and *Ccl2* in 3T3-L1 mouse cells (undifferentiated preadipocytes and differentiated adipocytes) treated with 300 μg/mL AGE, 1 μg/mL HMGB1, or 100 ng/mL S100B for 24 h. The levels of the mRNAs were measured via real-time RT-PCR using *rat insulinoma gene* (*Rig*)/*ribosomal protein S15* (*RpS15*) as an endogenous control. Data are expressed as mean ± SE for each group (*n* = 4). The statistical analyses were performed using Student's *t*-test vs. No addition.

#### *2.3. Down-Regulation of RAGE Attenuated the Increases of IL-6 and CCL2 in Adipocytes Treated with Small Interfering RNA (siRNA) for RAGE*

In order to see the mechanism of HMGB1- and AGE-induced gene expression of *IL-6* and *CCL2*, *RAGE* gene was knocked down by RNA interference. The expression of *IL-6* and *CCL2* was significantly increased by the addition of HMGB1 and AGE even in the presence of scrambled RNA. In contrast, introduction of small interfering RNA (siRNA) for RAGE (*siRAGE*) clearly inhibited the HMGB1- and AGE-induced increases of mRNAs for *IL-6* and *CCL2* in SW872 human adipocytes (Figure 4; *P* = 0.2638 [No addition vs. HMGB1 in *IL-6*], *P* = 0.0744 [No addition vs. AGE in *IL-6*], *P* = 0.2559 [No addition vs. HMGB1 in *CCL2*], and *P* = 0.5541 [No addition vs. AGE in *CCL2*]).

We also measured the concentrations of IL-6 and CCL2 in the RAGE-knocked-down SW872 cell culture medium via enzyme-linked immunosorbent assay (ELISA). The concentrations of IL-6 and CCL2 were significantly increased in response to HMGB1 and AGE in scrambled RNA-introduced cell culture medium. In contrast, the introduction of siRAGE significantly attenuated the HMGB1- and AGE-induced increases of IL-6 and CCL2 in the medium (Figure 5).

#### *2.4. Up-Regulation of IL-6 and CCL2 by Lipopolysaccharide (LPS) in Adipocytes*

Recent reports indicated that PE/HDP is also induced by lipopolysaccharide (LPS) [32] and that RAGE mediates LPS signaling and acts as an LPS receptor [33–38]. Thus, we tested whether LPS up-regulate gene expression of *IL-6* and *CCL2* in human SW872 adipocytes. We added 10 ng/mL LPS in SW872 culture medium, incubated for 24 h, and the expression of *IL-6* and *CCL2* was analyzed via real-time RT-PCR. As shown in Figure 6, mRNAs of *IL-6* and *CCL2* were significantly up-regulated by the addition of LPS.

**Figure 4.** Effects of siRNA against *RAGE* on HMGB1- and AGE-induced gene expression of *IL-6* and *CCL2*. SiRNA for *RAGE* was transfected into SW872 cells and the cells were incubated with HMGB1 or AGE for 24 h. The levels of *IL-6* and *CCL2* mRNA were measured via real-time RT-PCR using β*-actin* as an endogenous control. Data are expressed as mean ± SE for each group (*n* = 4). The statistical analyses were performed using Student's *t*-test vs. No addition.

**Figure 5.** Effect of siRNA against RAGE on the HMGB1- and AGE-induced expression of IL-6 and CCL2. SiRNA for RAGE was transfected into SW872 cells and the cells were incubated with HMGB1 or AGE for 24 h. The levels of IL-6 and CCL2 in the cell culture medium were measured via ELISA. Data are expressed as mean ± SE for each group (*n* = 4). The statistical analyses were performed using Student's *t*-test vs. No addition.

**Figure 6.** The mRNA levels of IL-6 and CCL2 in SW872 human adipocytes treated with 10 ng/mL lipopolysaccharide (LPS) for 24 h. The levels of the mRNAs were measured via real-time RT-PCR using β-actin as an endogenous control. Data are expressed as mean ± SE for each group (*n* = 4). The statistical analyses were performed using Student's *t*-test.

We next measured IL-6 and CCL2 in the LPS-stimulated SW872 cell culture medium and found that the levels of IL-6 and CCL2 in the LPS-stimulated SW872 culture medium were also elevated significantly (Figure 7).

**Figure 7.** The levels of IL-6 and CCL2 in culture medium of SW872 human adipocytes treated with 10 ng/mL LPS for 24 h. The levels of IL-6 and CCL2 in the cell culture medium were measured via ELISA. Data are expressed as mean ± SE for each group (n = 4). The statistical analyses were performed using Student's *t*-test.

#### *2.5. Down-Regulation of RAGE Attenuated the LPS-Induced IL-6 and CCL2 Increases in Adipocytes*

In order to confirm whether the mechanism of LPS-induced *IL-6* and *CCL2* up-regulation is also mediated by RAGE, *RAGE* gene was knocked down by RNA interference. The expression of *IL-6* and *CCL2* was significantly increased by the addition of LPS even in the presence of scrambled RNA. In contrast, introduction of *siRAGE* clearly inhibited the LPS-induced increases of mRNAs for *IL-6* and *CCL2* in SW872 human adipocytes (Figure 8).

We also measured the concentrations of IL-6 and CCL2 in the RAGE-knocked-down SW872 cell culture medium via ELISA. The concentrations of IL-6 and CCL2 were significantly increased in response to the addition of LPS in scrambled RNA-introduced cell culture medium. In contrast, the introduction of siRAGE significantly attenuated the LPS-induced increases of IL-6 and CCL2 in the medium (Figure 9).

**Figure 8.** Effects of siRNA against *RAGE* on the LPS-induced gene expression of *IL-6* and *CCL2*. SiRNA for *RAGE* was transfected into SW872 cells and the cells were incubated with 10 ng/mL LPS for 24 h. The levels of *IL-6* and *CCL2* mRNA were measured via real-time RT-PCR using β*-actin* as an endogenous control. Data are expressed as mean ± SE for each group (*n* = 4). The statistical analyses were performed using Student's *t*-test.

**Figure 9.** Effect of siRNA against RAGE on the LPS-induced expression of IL-6 and CCL2. SiRNA for RAGE was transfected into SW872 cells and the cells were incubated with LPS for 24 h. The levels of IL-6 and CCL2 in the cell culture medium were measured via ELISA. Data are expressed as mean ± SE for each group (*n* = 4). The statistical analyses were performed using Student's *t*-test.
