Research ArticleThe Effect and Mechanism of TLR9/KLF4 in FFA-InducedAdipocyte Inflammation

Cuizhe Wang ,1 Meixiu Zhang,1 Jinxiu Wu,1 Wei Li ,2 Xiaodan Ha ,1 Yajuan Gu ,3

Bo Han ,4 Jianxin Xie ,1 and Jun Zhang 1

1Shihezi University, School of Medicine, Shihezi, Xinjiang 832000, China2Shihezi University School of Medicine in the First Affiliated Hospital Clinical Laboratory, Shihezi, Xinjiang 832000, China3Shihezi University School of Medicine in the First Affiliated Hospital Department of Obstetrics and Gynecology, Shihezi,Xinjiang 832000, China4Shihezi University, School of Pharmacy, Shihezi, Xinjiang 832000, China

Correspondence should be addressed to Jianxin Xie; mayue850911@163.com and Jun Zhang; zhangjunyc@163.com

Received 15 August 2018; Revised 1 October 2018; Accepted 15 October 2018; Published 18 December 2018

Academic Editor: Michele T. Pritchard

Copyright © 2018 Cuizhe Wang et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective. Current research has reported that obesity is a chronic inflammatory state, which is closely related with excessiveaccumulation of free fatty acid, while the specific mechanism that high level of FFA causes inflammation is not very clear. Thus,our research intended to observe the high FFA effects on TLR9/KLF4 expression and the downstream inflammatory factors, toexplore the mechanism of inflammatory response suppressed by TLR9/KLF4. Methods. qRT-PCR and Western blot were usedto detect the mRNA and protein expression levels of TLR9, KLF4, and key inflammation-related factors. ELISA was used todetect the release level of inflammatory cytokines. The triglyceride (TG) and glucose (GLU) testing cassettes were used to detectthe TG and GLU levels in culture medium. Results. In the omental tissue of obese individuals (OB), we found that TLR9, KLF4,mRNA, and the protein expression levels were lower than those of the normal weight control (NC) group. Similarly, in theomental tissue of high-fat diet (HFD) rats, we found that the mRNA expression levels of TLR9 and KLF4 were lower than thoseof the normal diet control group. In mature adipocytes, we found that KLF4 played an important anti-inflammatory role;moreover, PA can promote the development of inflammation by inhibiting KLF4 expression; TLR9 has a positiveregulation function on KLF4 expression, but unrelated to PA. Conclusions. TLR9/KLF4 is involved in regulating FFA-inducedadipocyte inflammation.

1. Introduction

Obesity refers to the excessive accumulation or abnormaldistribution of fat, usually associated with weight gain, whichhas become a worldwide health problem that can lead tocardiovascular disease, hypertension, diabetes, and othermetabolic diseases [1, 2]. Current research has reported thatobesity is a chronic inflammatory state, which is closelyrelated with excessive accumulation of free fatty acid (FFA)due to lipolysis of adipocytes [1, 3]. The excessive accumu-lation of FFA will cause the activation of inflammatorysignaling pathways, finally causing cell dysfunction [4]. Todate, the specific mechanism that high level of FFA causesinflammation is not very clear.

Kruppel-like factors (KLFs), as a transcription factorfamily, are composed of 17 members with zinc finger struc-ture, widely involving cell proliferation, differentiation, andembryonic developmental regulation [5]. KLF4, originallyseparated from the gastrointestinal tract, is one of thetranscriptional regulation factors of adipocyte differentiation[6]. In recent years, KLF4 has involved the regulation effect ofvarious chronic inflammatory responses. In vascular endo-thelial cell, KLF4 combined with the activity subunit P65 ofnuclear transcription factor κB (NF-κB), which caused theinhibition of the combination of P65 and vascular cell adhe-sion molecule 1 (VCAM1) to play an anti-inflammatory role[7]. Recently, it was reported that the overexpression of KLF4increased the M2 macrophage (anti-inflammation) marker

HindawiMediators of InflammationVolume 2018, Article ID 6313484, 10 pageshttps://doi.org/10.1155/2018/6313484

protein expression, while it decreased M1 macrophage(inflammation) marker protein expression. Moreover,KLF4-deficient macrophages exhibited lower ability toperform fatty acid oxidation [8]. In J774a cells, KLF4 overex-pression reduced the expression of MCP-1 [8]. In adipocytes,whether KLF4 plays an important role in FFA inducedinflammatory response has not been reported yet.

Toll-like receptors (TLRs) play an important role ininflammatory signaling pathways of congenital immuneresponse. Latest literature reported that TLR9 gene knockoutmice with a high-fat diet had more visceral fat accumulationand released inflammatory factors, such as IL-6, MCP-1and TNF-α [9]. In human lung epithelial cell, TLR9raised KLF4 expression through MYD88/SRC pathwayto promote the release of anti-inflammatory factor IL-10[10]. In adipocytes, whether TLR9/KLF4 plays an anti-inflammatory role in FFA-induced inflammatory responsehas not been reported.

Therefore, on the basis of constructing high-fat diet-induced obese rat model, studying the human omentaladipose tissue, and culturing adipocytes in vitro, our researchintended to explore the molecular mechanism of the inhibi-tion effect of TLR9/KLF4 on FFA-induced inflammatoryresponse of adipocytes, which can help to elucidate themolecular mechanism of obesity initiating inflammation.

2. Materials and Methods

2.1. Source of Tissue Samples. From January to December2014, 95 individual participants were enrolled with agesbetween 20 and 90 years old in the People’s Hospital ofXinjiang Uygur Autonomous Region for physical examina-tion and evaluation of dyslipidemia. They were dividedinto two groups: the normal control group (NC, n = 50,18.0 kg/m2≤BMI≤ 23.9 kg/m2) and the obese group (OB,n = 45, BMI≥ 28 kg/m2). Thirty-two Wistar male rats pro-vided by the Centers for Disease Control in Xinjiang UygurAutonomous Region were fed in the Center for ExperimentalAnimals of Shihezi University. The rats were divided into twogroups: high-fat diet (HFD) group (n = 16) and normal dietcontrol group (n = 16). This study was approved by themedical ethics committee (no. 2014LL22), and the partici-pants all signed an informed consent.

2.2. Cell Lines and Plasmid. The 3T3-L1 cell lines were boughtfrom the Cell Center of Basic Medical Institute of the ChineseAcademy of Medical Sciences. pIRES2, pIRES2-KLF4, TLR9lentiviral vector, and siRNA of TLR9 and KLF4 were boughtfrom Gemma Pharmaceutical Technology.

2.3. Real-Time PCR. Real-time PCR was performed usingSYBR Premix Ex Taq (Takara) on a 7500 Real-Time PCRSystem (Applied Biosystems, Foster City, CA). Primersequences are listed in Table S1, using GAPDH or β-actinas an internal control.

2.4. Cell Culture and Differentiation. The 3T3-L1 cells werecultured in DMEM supplemented with 10% fetal bovineserum and antibiotics. Culturing preadipocyte 3T3-L1 was

done to differentiate into mature adipocytes which isidentified by oil red O.

2.5. Transfection Assays. All transient transfection andadenoviral overexpression procedures were performed in3T3-L1 cell line. For overexpression, 3T3-L1 cells wereinfected with the empty control virus or the lentiviruscarrying the mouse TLR9 gene and the pIRES2-ZsGreen1or pIRES2-KLF4-ZsGreen1. Transient transfection assayswere done with lipo2000 transfection reagent (Invitrogen,USA). For downexpression, 3T3-L1 cells were infected withsmall interference RNA.

2.6. Western Blot. The total protein was analyzed bysodium dodecyl sulphate-polyacrylamide gel electrophore-sis (SDS-PAGE). Membranes were blocked and incubatedat 4°C overnight with antibodies. Then, secondary antibodywas incubated for 2 h at room temperature. Proteins werevisualized using the enhanced chemiluminescence (ECL)detection system (FluorChem HD2, USA).

2.7. Participant Consent and Ethics Statement. All partici-pants provided informed and voluntary consent prior toenrolment in the study. This consent included understandingthat clinical information and biological samples would beused for research. The consent form and ethical approvalwere provided by the Medical Ethics Committee at the FirstAffiliated Hospital, Shihezi University School of Medicine(reference number 2014LL22).

2.8. Statistical Analysis. SPSS (version 13.0, SPSS Inc.,Chicago, IL, USA) was used for data analysis. Student’st-test and rank sum test were used to compare thegroups. Correlation analysis was performed using thePearson method, and a P value < 0.05 was defined as sta-tistically significant.

3. Results

3.1. The mRNA and Protein Expression Levels of TLR9/KLF4and Inflammation-Related Factors in the Omental AdiposeTissue of Individual Participants. The clinical characteristicsof the individual participants in the NC and OB groups wereshowed in Tables S2 and S3. In the OB group, the mRNA andprotein expression levels of TLR9 and KLF4 were signifi-cantly lower than those in the NC group (P < 0 05), andAPN was lower than the NC group. The mRNA and proteinexpression levels of subunit p65 of NF-κB, TNF-α, and IL-6in the OB group were significantly higher than those in theNC group (P < 0 05) (Figures 1(a)–1(c)). In the OB group,the mRNA expression level of TLR9 was significantly andpositively correlated with HDL and KLF4 (P < 0 05); themRNA expression level of KLF4 was significantly and nega-tively correlated with BMI, TG, and TNF-α (P < 0 05)(Figure 1(d)).

3.2. The mRNA Expression Level of Inflammatory-RelatedFactors and the Correlation of FFA, TLR9/KLF4, andInflammatory-Related Genes in the Omental Adipose Tissueof Rats. Construction of the obesity animal model was

2 Mediators of Inflammation

shown in Figure S1 and Table S4. On the basis of obesitymodel, we investigated the mRNA expression levels ofTLR9/KLF4 and downstream inflammatory-related factors.The mRNA expression levels of TLR9 and KLF4 in the

HFD group were lower than those in the NC group,whereas the mRNA expression levels of TNF-α and IL-6in the HFD group were higher than those in the NCgroup (Figure 2(a)).

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Figure 1: The mRNA and protein expression levels of TLR9, KLF4, and key factors of omental adipose tissue. (a) mRNA expression level;(b) protein expression level; (c) gray scale scanning result; (d) the correlation of TLR9/KLF4 and blood lipid level/key factors ofinflammatory signaling pathways in the omental adipose tissue of the OB group. t-test, ∗P < 0 05, ∗∗P < 0 01; the difference wasstatistically significant; Pearson correlation analysis, P < 0 05; the difference was statistically significant.

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3Mediators of Inflammation

The FFA level was significantly and negatively correlatedwith TLR9 mRNA expression level (P < 0 05) and signifi-cantly and positively correlated with TNF-α (P < 0 05);KLF4 was significantly and positively correlated with TLR9(P< 0.05) and was negatively correlated to the subunit P65of NF-κB (Figure 2(b)).

3.3. Culture and Differentiation of 3T3-L1 Adipocytes. The3T3-L1 cell is fusiform and lipid droplet does not exist. On

differentiation by the 8th day, there were more than 90%mature adipocytes, gathering more lipid droplet. The oil redO staining results showed that the lipid droplet was dyedred. Hematoxylin stain results showed that the nuclear wasdyed purple (Figure 3(a)).

3.4. The Effects on Downstream Inflammatory Factors afterDownregulation of KLF4. After KLF4 interference for 24hand 48h, the KLF4 mRNA expression level was significantly

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Figure 3: The regulatory effect of KLF4 on downstream inflammatory factors. (a) The differentiation of 3T3-L1 adipocytes; (b) the mRNAand protein expression level of KLF4 after KLF4 interference for 24 h and 48 h; (c) detecting the protein expression level of p-P65 afterdownregulation of KLF4; (d) detecting the mRNA expression levels of P65, IL-6, MCP-1, TNF-α, and APN after downregulation of KLF4;(e) release levels of IL-6, MCP-1, TNF-α, and APN after downregulation of KLF4; (f) mRNA and protein expression levels of KLF4 afterpIRES2-KLF4 plasmid was transfected into mature adipocytes after 24 h and 48 h; (g) detecting the protein expression level of p-P65 afterupregulation of KLF4; (h) mRNA expression levels of P65, IL-6, MCP-1, TNF-α, APN, and IL-1β; (i) detecting the release levels of IL-6,MCP-1, TNF-α, and APN after upregulation of KLF4. t-test, ∗∗P < 0 01, ∗P < 0 05; the difference has a statistical significance. The effectson KLF4 expression after upregulation of TLR9.

4 Mediators of Inflammation

lower than the si-control and si-NC groups (P < 0 05). AfterKLF4 interference for 24h, the protein expression level ofKLF4 was also lower than the si-control and si-NC groups(Figure 3(b)). Next, we examined the mRNA expression leveland release level of inflammation-related factors after KLF4interference for 24 h. The results showed that the proteinexpression level of p-P65 was significantly lower than thatof the Ad-NC group (Figure 3(c)), and the mRNA expressionlevels of IL-6 and MCP-1 were significantly increasedcompared with those of the si-control group (P < 0 01), whilethe mRNA expression level of anti-inflammatory factorAPN was significantly decreased compared with that ofthe si-control group (P < 0 05) (Figure 3(d)). After KLF4interference, the release level of MCP-1 was also significantlyincreased (P < 0 01) (Figure 3(e)).

3.5. The Effects on Downstream Inflammatory Factors afterUpregulation of KLF4. After pIRES2-KLF4 plasmid wastransfected into mature adipocytes after 24 h and 48 h, themRNA expression levels of KLF4 were both significantlyincreased (P < 0 05), and in the 48 h transfection, the KLF4mRNA expression level was significantly higher than that inthe 24h transfection group (P < 0 05). Moreover, the KLF4protein expression level was also significantly increased inthe 48 h transfection group (Figure 3(f)). Therefore, weinvestigated the inflammation-related factor expression andrelease level in the group of 48 h transfection of KLF4plasmid. The results showed that the protein expression levelof p-P65 was significantly lower than that of the Ad-NCgroup (Figure 3(g)), and the mRNA expression levels ofinflammatory-related factors, such as P65, TNF-α, andIL-1β, were significantly lower than those of the Ad-NCgroup (P < 0 05), while the mRNA expression level ofanti-inflammatory factor APN was significantly higherthan that of the Ad-NC group (P < 0 05) (Figure 3(h)). Afterthe upregulation of KLF4, the release level of MCP-1 wassignificantly decreased (P < 0 05) (Figure 3(i)).

3.6. The Effects on KLF4 Expression after Upregulation ofTLR9. First, the MO1 numbers 0.3, 3, and 10, respectively,were infected into mature adipocytes for 72 h, 96 h, and120h. The results showed that the mRNA expression levelof TLR9 was highest when the MO1 was 3 and infected for96 h (Figure 4(a)). Therefore, we examined the TLR9 regula-tion on KLF4 under this condition. The results showed thatthe number of adipocytes has no significant difference com-pared with the mock and negative control group after TLR9overexpression. Moreover, TLR9 mRNA and protein expres-sion levels were both significantly higher than the mock andnegative control group after TLR9 overexpression (P < 0 01).After upregulation of TLR9, the mRNA and protein levels ofKLF4 were significantly higher than those of the mock andnegative control groups (P < 0 01) (Figures 4(c) and 4(d)).Compared with the mock and negative control groups,upregulated TLR9 can significantly decrease the mRNAexpression levels of P65 and MCP-1 (P < 0 05), while signif-icantly increase the expression level of APN (P < 0 01)(Figure 4(e)).

3.7. The Effects on KLF4 Expression after Downregulation ofTLR9. The negative interference fragment with red fluores-cence was transfected into mature adipocytes, and theconcentration gradients were 10, 50, and 100nmol/L. Then,we observed the transfection efficiency in 24 h and 48 h.The results showed that with the concentration increasing,the transfection efficiency was also increased, and thefluorescence intensity in the 48h transfection group wassignificantly higher than that in the 24 h group. When theconcentration of interference fragment was 50 nmol/L andthe interference time was 48 h, fluorescence intensity hadreached 80-90% (Figure 4(f)). Next, we screened the bestinterference fragment from three TLR9-siRNA interferencefragments, and the results showed that TLR9-siRNA-B01had the highest efficiency (Figure 4(g)). The number ofadipocyte has no significant difference compared with themock and negative control groups after TLR9-siRNA-B01fragment transfection (Figure 4(h)). Moreover, the mRNAand protein expression levels of TLR9 were significantlylower than those of the mock and negative control groupsafter TLR9-siRNA-B01 fragment transfection (P < 0 05).The mRNA and protein expression levels of KLF4 were sig-nificantly lower than those of the mock and negative controlgroups after downregulation of TLR9 (Figures 4(i) and 4(j)).Downregulated TLR9 can significantly increase the mRNAexpression levels of subunit P65 of NF-κB, IL-6, andMCP-1 (P < 0 05), while the APN is significantly lower(P < 0 01). Overexpressing KLF4 while knocking downTLR9, the mRNA expression levels of IL-6 and MCP-1were significantly descended (P < 0 05), while the mRNAexpression level of APN was significantly increased(P < 0 01) (Figure 4(k)).

3.8. Under Different Concentrations of PA Stimulation, theExpression Levels of TLR9/KLF4 and InflammatoryCytokines Were Detected. Selecting different concentrationsof saturated fatty acid PA (0, 0.2, 2, 20, 100, and 200μM)stimulates adipocytes for 48 h to detect the mRNA andprotein expression levels of TLR9/KLF4. We found that withthe increase of PA concentration, the protein expression levelof TLR9 had no statistical significance, while the level ofKLF4 was increased at first and then decreased. When thePA concentration is 20μM, the KLF4 expression level wasthe highest, and the difference was statistically significantcompared with the NC group (P < 0 05). Under the 100μMand 200μM PA stimulation, the mRNA and proteinexpression levels of KLF4 were significantly decreased com-pared with those of the 20μM PA stimulation (P < 0 05)(Figure 5(a)). In the 20μMPA stimulation group, the mRNAexpression levels of IL-6, MCP-1, and APN and the releaselevel of IL-6 were significantly higher than those in the0μM PA stimulation group. In the 200μM PA stimulationgroup, the mRNA expression levels of P65, IL-6, MCP-1,and IL-1β and the release levels of IL-6 and MCP-1 weresignificantly higher than those in the 0μM PA stimulationgroup. In the 200μM PA stimulation group, the mRNAexpression level of IL-6 and the release levels of IL-6 andMCP-1 were significantly higher than those in the 20μMPA stimulation group, while the mRNA expression level of

5Mediators of Inflammation

APN was significantly lower than that of the 20μM PA stim-ulation group, and the above differences were all statisticallysignificant (P < 0 05) (Figure 5(b)).

3.9. Detecting the Expression of Downstream InflammatoryFactors under the 20μM PA Stimulation andDownregulated KLF4. Under the 20μM PA stimulation withdownregulated KLF4, the mRNA and protein expressionlevels of KLF4 were significantly decreased (P < 0 01) (FigureS2). Next, we investigated the expression of downstreaminflammatory factors. We found that under the 20μM PA

stimulation, downregulated KLF4 increased the mRNAexpression levels of MCP-1 and IL-1β and the release levelof IL-6 (P < 0 05), while it inhibited the mRNA expressionlevel of the APN (P < 0 01) (Figures 5(c) and 5(d)).

3.10. Detecting the Expression of Downstream InflammatoryFactors under the 200μM PA Stimulation and UpregulatedKLF4. Under the 200μM PA stimulation with upregulatedKLF4, the mRNA and protein expression levels of KLF4were significantly increased (P < 0 01) (Figure S3). Next,we examined the expression of downstream inflammatory

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Figure 4: Detection of TLR9 regulation on KLF4 and key factors. (a) TLR9 mRNA expression level in MOI = 0.3, 3, and 10 and time = 72, 96,and 120 h, respectively; (b) oil red O staining results of the mock, negative control, and virus infection groups; (c, d) upregulated TLR9,detecting theTLR9 and KLF4 mRNA and protein expression levels; (e) upregulated TLR9, detecting the mRNA expression level of P65, IL-6, MCP-1, IL-1β, and APN; (f) the fluorescence in concentration = 10, 50, and 100 nmol/L and time = 24 h and 48 h; (g) the interferenceefficiency of three different TLR9-siRNA; (h) oil red O staining results of the mock, negative control, and TLR9-siRNA groups; (i, j)downregulated TLR9, detecting the mRNA and protein expression levels of TLR9 and KLF4. t-test, ∗∗P < 0 01, ∗P < 0 05; the differencehas a statistical significance; (k) overexpressing KLF4 while knocking down TLR9, detecting the mRNA expression level of IL-6, MCP-1,TNF-α, and APN. ANOVA test, ##P < 0 01, #P < 0 01 compared with si-NC; ∗∗P < 0 01, ∗P < 0 05 compared with si-TLR9.

6 Mediators of Inflammation

factors. We found that under the 200μM PA stimulation,upregulated KLF4 could significantly suppress the expressionlevels of subunit P65 of NF-κB, TNF-α, and IL-1β, while itincreased the mRNA expression level of APN (P < 0 05)(Figures 5(e) and 5(f)).

3.11. Detecting the KLF4 Effects on Glycolipid MetabolismAbility. In culture medium, upregulated KLF4 could signifi-cantly suppress the level of TG and GLU (P < 0 05), anddownregulated KLF4 could significantly increase the levelof TG (P < 0 05). Under the 20μM PA stimulation,

GAPDH

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(f)

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+−−−

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l (fo

ld)

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(h)

Figure 5: High FFA promotes the expression level of inflammation factors by downregulating KLF4. (a) Different concentrations of PA wereused to stimulate cells to detect the protein expression levels of TLR9/KLF4; (b) the mRNA expression levels of P65, IL-6, MCP-1, TNF-α,APN, and IL-1β and the release expression levels of IL-6 and MCP-1 under the 20 μM and 200 μM PA stimulation; (c) detecting themRNA expression levels of P65, IL-6, MCP-1, TNF-α, APN, and IL-1β after 20 μM PA stimulation and si-KLF4; (d) detecting the releaseexpression levels of IL-6, MCP-1, TNF-α, and APN after 20 μM PA stimulation and si-KLF4; (e) detecting the mRNA expression levels ofP65, IL-6, MCP-1, TNF-α, APN, and IL-1β after 200 μM PA stimulation and KLF4 transfection; (f) detecting the release expression levelsof IL-6, MCP-1, TNF-α, and APN after 200μM PA stimulation and KLF4 transfection; (g, h) the levels of TG and GLU after 20μM PAstimulation and si-KLF4. t-test, ∗∗P < 0 01, ∗P < 0 05, #P < 0 05; the difference has a statistical significance.

7Mediators of Inflammation

downregulated KLF4 could significantly increase the level ofTG and GLU (P < 0 05) (Figures 5(g) and 5(h)).

4. Discussion

Obesity is a state of chronic inflammation and is closelyrelated to insulin resistance, type 2 diabetes, and othermetabolic diseases [11]. The accumulation of FFA plays animportant role in the inflammatory response. Therefore,investigating the specific molecular mechanism of FFA-induced inflammatory response of the adipose tissue willprovide theoretical basis for the treatment of obesity andmetabolic disorders. Thus, on the basis of constructinghigh-fat diet-induced obese rat model, the human omentaladipose tissue, and culturing adipocytes in vitro, our researchintended to explore the molecular mechanism of the inhibi-tion effect of TLR9/KLF4 on FFA-induced inflammatoryresponse of adipocytes.

4.1. TLR9 Can Promote KLF4 Expression to Exert Anti-Inflammatory Effect. Existing literature reported that KLF4,as a transcription factor, plays an anti-inflammatory role inthe different cell types, such as vascular endothelial cell, lungepithelial cell, and macrophage [5–8]. Whether KLF4 playsan anti-inflammatory role in adipocytes has not beenreported yet. Our results showed that downregulated KLF4promoted the expression of inflammatory cytokines IL-6and MCP-1. Upregulated KLF4 inhibited the expression ofinflammatory cytokines IL-1β and MCP-1. The above resultssuggested that KLF4 played an anti-inflammatory role inadipocytes. Existing literature showed that TLR9 couldinhibit inflammatory reaction in adipose tissues, colitis tis-sues, intestinal tissues, and human lung epithelial cells [12].Our results showed that downregulated TLR9 significantlydecreased the mRNA and protein expression levels of KLF4and anti-inflammatory factor APN, while it significantlypromoted the inflammatory factor expression. These resultssuggest that TLR9 plays an anti-inflammatory role byincreasing KLF4 expression.

4.2. High Concentration of PA Inhibits KLF4, While It Has NoEffects on TLR9. Due to the important role of FFA in obesity-induced inflammation, we then investigated the FFA effectson TLR9/KLF4. In individual participants, we found thatthe levels of TG and LDL were significantly higher in theOB group, the mRNA and protein expression levels ofKLF4 were significantly lower in the OB group comparedwith the NC group, and KLF4 was significantly and nega-tively correlated with TG. The neutral fat including TG canbe decomposed into FFA that is a key factor of inflammation.The above results suggested that high concentration of FFAmight inhibit the expression level of KLF4. In the NC group,the TG level was lower, while the expression level of KLF4was higher, which suggested that low concentration of FFAmight promote the expression level of KLF4. Recent researchalso put forward that KLF4 knockout could inhibit the lipidintake and fatty acid oxidation ability in macrophagocyte[8]. Moreover, the elongation of long-chain fatty acid familymember 6 (Elovl6) can suppress the KLF4 expression [13].

Thus, we investigated the expression level of KLF4 andinflammatory factors under the different concentrations ofPA stimulated in adipocytes. The results showed that underthe low concentration of PA stimulation, the high expres-sion level of KLF4 could improve the glucolipid metabo-lism ability. However, under high concentrations of PAstimulation, the lower expression level of KLF4 could pro-mote the expression of inflammatory factors IL-6, IL-1β,and MCP-1. The above results suggest that high PA con-centration promotes the expression of inflammatory factorby inhibiting KLF4.

In the adipose tissue of the OB group of individual partic-ipants, TLR9 mRNA and protein expression levels weresignificantly lower than those of the NC group, and themRNA expression level of TLR9 was significantly and posi-tively correlated with HDL and KLF4. Meanwhile, in theadipose tissue of the HFD group of rats, the mRNA expres-sion level of TLR9 was lower than that of the NC groupand was significantly and negatively correlated with FFAwhile positively correlated with KLF4. The above resultssuggest that abnormal blood lipid level was significantlycorrelated with TLR9 and KLF4 expression in the adiposetissue. In adipocyte culture, we found that TLR9 positivelypromoted KLF4 expression, and while under differentconcentrations of PA, the TLR9 expression level has nosignificant difference. The above results suggest that highPA concentration that inhibits KLF4 expression was not viaTLR9. So, how does the PA inhibit KLF4? DNA methylationis an important means of regulation of eukaryotic geneexpression and a bridge that connects environment changeand cellular response. Existing literature reported thathigh-fat diet can lead to many metabolism-related genemethylation status [14, 15]. Whether PA inhibits KLF4expression due to the gene methylation status changingshould be studied further.

In vivo, we found that TLR9 was significantly negativelycorrelated with FFA, while PA did not regulate TLR9in vitro, which is inconsistent. The reason we suspect is thedifferent kinds of FFA. There are 37 kinds of FFA, andTLR9 expression may be correlated with other fatty acidbut not PA, such as oleic acid and arachidonic acid. The spe-cific mechanism needs to be further studied.

4.3. KLF4 Upregulation of the Expression Level of APN. APN,specifically secreted by adipocytes, is an anti-inflammatorymolecule that can suppress immune response and atheroscle-rosis and is negatively correlated with varieties of metabolicdiseases caused by obesity [16]. Our results showed thatcompared with the NC group, the expression level of APNwas lower in the OB group. In adipocytes, high PA concen-tration inhibited APN expression. Upregulated KLF4 couldsignificantly increase the level of APN, and downregulatedKLF4 could significantly decrease the level of APN. More-over, under the high concentration of PA stimulation, KLF4overexpression could significantly increase the APN expres-sion. Under the low concentration of PA stimulation, KLF4downexpression could significantly reduce APN expression.The above results indicated that high levels of FFA inhibitedthe expression level of APN through downregulating KLF4,

8 Mediators of Inflammation

eventually leading to the inflammatory reaction. KLF4 as atranscriptional regulation factor, and whether KLF4 directlyregulated APN gene expression needs further research.

In summary, our research has explored the molecularmechanism of the inhibition effect of TLR9/KLF4 on FFA-induced inflammatory response of adipocytes, which canhelp to elucidate the molecular mechanism of obesityinitiating inflammation and to provide a new experimentalbasis. Although our research found that FFA promotedinflammation by inhibiting TLR9/KLF4 in adipocytes, wedid not exclude the role of key factors on the process ofinflammation. Therefore, in the future, we still need tofurther explore their specific molecular mechanism.

Data Availability

The data of qRT-PCR, Western blot, and ELISA used tosupport the findings of this study are available from thecorresponding author upon request.

Conflicts of Interest

The authors declare that they have no competing interests.

Authors’ Contributions

Cuizhe Wang and Meixiu Zhang contributed equally tothe paper.

Acknowledgments

We thank Dr. Han, from the School of Pharmacy of XinjiangShihezi University, for the assistance with providing the tra-ditional Uygur medicine, Turkish galls, to finish our research.This work was supported by the Natural Science Foundationof China (nos. 81360142 and 81660137), the Applied BasicResearch Project of Xinjiang Corps (2015AG016), the Pro-jects of Shihezi University (ZZZC201701A), and the StudentResearch Training Program (SRP2018074).

Supplementary Materials

Table S1: primer sequences of fragments. Table S2: compari-son of subject metrics and biochemical parameters betweenthe NC and the OB group in the individuals. Table S3: com-parison of biochemical indexes and inflammatory factor levelbetween the NC and the OB group in the individuals. TableS4: the comparison of blood glucose, lipid, adipocytokines,and inflammatory factor level in the rat model of obesity.Figure S1: construction of obese animal model. The rats weremonitored for 10 weeks while being fed normal diet andhigh-fat diet. (A) Body weight (n = 8 rat per group). (B)The appearance of the rat. Scale bar: 1 cm. (C) The visceralfat mass and liver appearance. Scale bar: 1 cm. t-test, valuesare given as the mean± SD. ∗P < 0 05, ∗∗P < 0 01 comparedwith the NC group. Figure S2: the expression levels of KLF4after 20μM PA stimulation and si-KLF4. (A) mRNA expres-sion level of KLF4. (B) Protein expression level of KLF4.t-test, ∗∗P < 0 01, ∗P < 0 05; the difference has a statisticalsignificance. Figure S3: the expression levels of KLF4 after

200μM PA stimulation and KLF4 transfection. (A) mRNAexpression level of KLF4. (B) Protein expression level ofKLF4. t-test, ∗∗P < 0 01, ∗P < 0 05; the difference has a statis-tical significance. (Supplementary Materials)

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