The PCSK9 Involves in the Dyslipidemia of Ovariectomized Mice and Its Mechanism

Dan Zhao Sichuan University West China School of Basic Medical Sciences and Forensic Medicine Xue-qin Zhang Sichuan University West China School of Basic Medical Scinences and Forensic Medicine Wen-jing Guo Sichuan University West China School of Basic Medical Sciences and Forensic Medicine Zhi-hui Cui Sichuan University West China School of Basic Medical Sciences and Forensic Medicine Yi-cheng Wang Sichuan University West China of Basic Medical Sciences and Forensic Medicine Yong-dan Ma Sichuan University West China School of Basic Medical and Forensic Medicine Li-xue Zhang Sichuan University West China School of Basic Medical Sciences and Forensic Medicine Jin-hu Zhang DM Wayanad Institute of Medical Sciences Department of Forensic Medicine Li Nie Sichuan University West China School of Basic Medical Sciences and Forensic Medicine Limin Yue (  yuelimin@scu.edu.cn ) Sichuan University West China School of Basic Medical Sciences and Forensic Medicine https://orcid.org/0000-0002-7631-5733


Introduction
One of the main ovarian functions is the synthesis and secretion of female estrogen (E2) which maintains the structure and fuctions of reproductive system, also involved in regulating the functions of other systems and organs, such as cardiovascular system, urinary system, nervous system and so on [1].
Ovarian failure in menopausal women inevitablyleads to a sharp decrease of E2 which causes metabolism syndrome including obesity, cardiovascular disease, osteoporosis, anxiety etc.The disorders of lipid metabolism after menopause usually encompass total cholesterol (TC), low-density lipoprotein cholesterol (LDL-c) and triglycerides (TG) increased and high-density lipoprotein cholesterol (HDL-c) decreased [2], in which LDL-c is an important risk factor of atherosclerosis and cardiovascular disease [3].
In recent years, the consumption of a high-fat diet (HFD) has been increasingly viewed as a signi cant factor for aggravating the disorder of lipid metabolism in menopause [4]. However, the mechanism of lipid metabolism disorders caused by estrogen de ciency among the women in menopause is still unclear.
Low-density lipoprotein receptor (LDLR) on the hepatocyte membranes plays an important role in regulating LDL-c in blood. LDL-c binding to LDLR is internalized by endocytosis under the participation of reticulin, then LDL-c dissociates from LDLR for cellular utilization oris degraded in the acidic environmentof intracellular lysosomes, while LDLR is transported back to the cell membrane for recycle [5]. Once the LDLR fails to recycle, the number of LDLRs in the cell membrane decreases, the level of LDL-C in blood will elevate. Proprotein convertase subtilisin/kexin type-9 (PCSK9) is a secreted protein rstly found in the liver a few years ago. PCSK9 may binds to the LDLR on cell membranes to form a complex, which hinders its recycling to lead to its degradation [6] so that the level of LDL-c in blood incrase. Therefore, PCSK9 is considered to be a negative regulator of cholesterol metabolism and is related to abnormal lipid metabolism and coronary heart disease [7]. PCSK9 inhibitors have been used in the treatment of hypercholesterolemia and mixed dyslipidemia. Clinical data has suggested that the elevation of PCSK9 is positively correlated with the increase of blood LDL-C in postmenopausal women [7]. Thus, it is not di cult to infer that there is an inevitable link among estrogen de ciency and PCSK9, as well as the lipid metabolism disorders in postmenopausal women.
In this research, wild-type(WT)C57BL/6 female mice and PCSK9 knockout PCSK9 −/− female mice were used to explore PCSK9 involves in the dyslipidemia of ovariectomized mice and its mechanism. At rst, we fed the mice with high-fat diet or normal diet for 4 weeks after ovariectomy, then we tested the mouse lipid metabolism pro les,PCSK9 in the blood and the expression of LDLR in liver, analyzed the correlation between PCSK9 and the lipid pro les. Futhermore, we treated the high-fat diet fed ovariectomized mice respectivelywith estrogen, PCSK9 inhibitor alone or the combination of the both for 2 weeks and checked the same items as the above. The purpose is to provide a new cluefor the diagnosis, treatment and prevention of postmenopausal lipid metabolism disorders.

Animal Experiments
All animal experiments were approved by the Sichuan University academic ethics and ethics committee and conducted in strict accordance with laboratory animal care and use program.
To clarify the changes of PCSK9 and lipid metabolism, as well as the relationship between both of them in ovariectomized mice, 20 10-week-old C57BL/6 female mice were divided into ovariectmized group and sham operation group. The half of two proups were fed with normal diet for 4 weeks, and the others were fed with high-fat diet for 4 weeks.Then all of them were checked E2, TC, TG, LDL-c, HDL-c and PCSK9 in the blood also we measured the PCSK9 and LDLR in their liver tissues.
To further verify PCSK9 is a important cause of dyslipidemia in ovariectomized mice, 8 10-week-old wildtype female mice (WT) and 8 10-week-old PCSK9 −/− female mice were ovariectomized and fed with highfat diet for 4 weeks, then, they were tested the same items as the above.
Next experiment was done in 20 female 10-week-old C57BL/6 ovariectomized female mice (SPF) which were fed with high-fat diet for 4 weeks, then they were divided into four groups, 5 animals per group. One group were given with estrogen along (OVCE group,1mg/kg/week), one group with PCSK9 inhibitor alone (OVCPi group, 3mg/kg/3d), one group with both estrogen and PCSK9 inhibitor for 2 weeks, and the last group as control. Two weeks later, all of the mice were tested as the above experiment.

Western Blot
Total protein extracts from the mouse livers were loaded on SDS-PAGE gels for western blot. Western blot was performed according to standard protocol. The antibodies of anti-LDLR (1:3000) and anti-PCSK9 (1:3000) were used. All experiments were repeated at least three times.

Examination of PCSK9 in the blood
The content of PCSK9 were tested by ELISA(R&D) according to the instructions of PCSK9 Elisa kit.

Lipid pro les analysis
Serum samples of the mice were analyzed with biochemical analysis kits (Nanjing Jiancheng Bioengineering Institute) to detected TC, TG, LDL-c and HDL-c.
Mouse tail genomic DNA extraction, DNA ampli cation and genotype identi cation of PCSK9-/mouse The Mouse tail genomic DNA were extracted by the Mouse tail genomic DNA extraction kit (APExBIO).
The mouse tail DNA ampli ed by PCR. The genotype identi cation by 2% agarose gel electrophoresis. The primer sequences used for PCR were as follows (Table 1)

Statistical analysis
The results of WB were analyzed by ImageJ software for grayscale analysis, and then all the data were statistically analyzed using Prism 5 software. Univariate ANOVA was performed according to the paired experiment design, and p<0.05 was considered statistically signi cant.

Results
Result 1: PCSK9 increased in the blood and liver of the ovariectomized mice, as well as lipid metabolism disordered in them, and there is positive relationship between their blood PCSK9 and LDL-c.
ELISA examining mouse blood PCSK9 showed that no matter whether the ovariectomized mice were given with normal diet or high-fat diet, their blood PCSK9 increased signi cantly ( Figure 1A. p<0.001, p<0.01), and it increased more in those with high-fat diet (93.6%) than those with normal diet (77.2%) ( Table 2). The examination of lipid pro les showed that the serum TC in the two groups of ovariectomized mice was both signi cantly increased (Figure 1.B1. p<0.05, p<0.01), and it increased in those with high-fat diet (50.4%) more than those with normal diet (23.2%) ( Table 2); The blood LDL-c of both also increased signi cantly (Figure 1.B2. p<0.01, p<0.05), in the same way, the normal diet mice increased more (68.6%) than those the high-fat diet mice (31.4%) ( A. PCSK9 in the blood increased in ovariectomized mice, B1-B4. Lipid pro les in the blood; C1-C2. The correlation analysis between PCSK9 and TC under the two diets; D1-D2. The correlation analysis between PCSK9 and LDL-c under the two diets; * represents p<0.05, ** represents p<0.01, *** represents p<0.001 IHC results showed that no matter whether the ovariectomized mice were given with normal diet or highfat diet, PCSK9 in their liver was signi cantly increased,while the expression of LDLR in the liver decreased (Figure 2.A). WB results was consistent with that of IHC (Figure 2.B1-B3, C1-C3).
Result3: There were no signi cant lipid disorders in PCSK9 -/ovariectomized mice To further prove PCSK9 in involved in lipid disorders in ovariectomized mice, PCSK9 −/− mice were used for experiment. The genotype identi cation of the mouse was as follows ( Figure 3A). Compared with the WT mice group, in which TC and LDL-c in the blood of were signi cantly increased in the ovariectomized mice (Figure 3.B1.B2 p<0.01, p<0.01); Compared with the PCSK9 −/− mice, there were no signi cant change of blood TC and LDL-c in the PCSK9 −/− ovariectomized mice (Figure 3. B1.B2). IHC results also showed that the expression of LDLR in the liver decreased in the ovariectomized WT mice, but not in the ovariectomized PCSK9-/mice ( Figure 3C). WB results were consistent with the IHC results. (Figure  3.D1.D2).
Result4: Estrogen and PCSK9 inhibitor improved the lipid disorders in ovariectomized mice.
After ovariectomized mice were treated respectively with estrogen or PCSK9 inhibitor, PCSK9, TC and LDL-c in the blood decreased signi cantly ( Figure 4A, B1, B2, p<0.01, p<0.05, p<0.05). IHC results showed PCSK9 decreased while LDLR increased in the mouse liver ( Figure 4C). WB results also showed LDLR increased in the liver( Figure 4D1,D3).; When the ovariectomized mice were treated with PCSK9 inhibitor, PCSK9, TC and LDL-c in the blood decreased( Figure 4A, B1, B2, p<0.05, p<0.05, p<0.01). IHC results showed PCSK9 decreased and LDLR increased in the liver ( Figure 4C). WB results were consistent with the IHC results ( Figure 4D1-D3). However, PCSK9, TC and LDL-c in the blood showed no signi cant differences among the ovariectomized mice treated with estrogen or PCSK9 alone or combined ( Figure  4A, B1, B2). PCSK9 and LDLR in the liver also showed no signi cant differences among the three group ( Figure 4.C, D1-D3).

Discussion
In order to prove estrogen de ciency causing high expression PCSK9 and its relation to lipid metabolism disorders among the women in menopause, we rstly used ovariectomized mice to test their serum PCSK9 and lipid pro les. The results suggest that the ovariectomized mice had both lipid disorders and increased PCSK9, which is consistent with some result in clinical studies that is there is an increase of PCSK9 in the circulating blood of postmenopausal women [8]. Ruscica et al tested serum PCSK9 in 227 premenopausal women and 460 postmenopausal women and found that PCSK9 was signi cantly increased after menopause and PCSK9 was positively correlated with the degree of arteriosclerosis Related [9]. Clinical studies have shown that lipid metabolism disturbed after menopause, which is characterized by TC and LDL-c increased, accompanied by TG little increased and HDL-c decreased [2]. Thailand's clinical study of 436 subjects (152 males, 143 menopausal women and 141 postmenopausal women) showed that serum PCSK9 of female postmenopausal increased signi cantly and was positively correlated with TC, LDL-c and BMI. PCSK9 increased the risk of cardiovascular disease [10]. This is consistent with our results. The results of Qin 's study showed that postmenopausal women's serum PCSK9 increased signi cantly, and it was positively correlated with total cholesterol and diastolic blood pressure of age. The results showed that PCSK9 is involved in the lipid metabolism disorders in menopausal women and our result have given another more evidence.
In order to further prove the role of PCSK9 in lipid metabolism disorders in ovariectomized mice, we compared the changes in lipid pro les between WT mice and PCSK9-/mice after ovariectomy, the results suggest that there is no obvious lipid metabolism disorder in PCSK9 −/− ovariectomized mice after ovariectomy. Clinical studies have shown that patients with PCSK9 gene deletion have increased the clearance of LDL and decreased LDL-c in plasma [11]. Jonathan C conducted a cohort study of people and found that a non-sense mutation of the PCSK9 gene led to plasma LDL-c decreased and the incidence of coronary heart disease decreased [12]. At the same time, it was also found in animal experiments that the expression of LDLR in mouse liver increased and LDL-c decreased in PCSK9 knockout mice [13]. Our previous experiments have also shown that lipid metabolism in ovariectomized mice is closely related to increased PCSK9. These studies combined with ours may prove that PCSK9 is one of the important causes of the lipid metabolism disorder in ovariectomized mice.
To investigate the effect of estrogen on PCSK9 expression, we tested the PCSK9 in the serum and liver of ovariectomized mice treated with estrogen. The results showed that PCSK9 both in the blood and liver decreased after the ovariectomized mice were treated with estrogen. Clinical studies have shown that estrogen in women is negatively correlated with PCSK9 and LDL-C [14]. Persson found High-dose ethinyl estradiol treatment in rats reduced the expression of PCSK9 while increased the expression of liver LDLR, but the mRNA level of LDLR decreased. The regulation of estrogen on liver LDLR may be completed by the post-transcriptional regulation of PCSK9 [15]. All these suggest that PCSK9 is regulated by estrogen.
Our results showed that when mice were ovariectomized, PCSK9 in the blood and liver increased; while when the ovariectomized mice were treated with estrogen, PCSK9 in the blood and liver decreased. All of the results indicate that the decrease of estrogen in ovariectomized mice is an important reason for the increase of PCSK9.
Above all, the increase of PCSK9 in mice ovariectomized as estrogen de ciency causes the decrease of LDLR in the liver, nally leads to disorders of lipid metabolism, which suggests that PCSK9 plays an important role in the dyslipidemia of ovariectomized mice, Therefore, PCSK9 inhibitor would be helpful for for improving the lipid metabolism disorder and reducing the incidence of cardiovascular disease in menopausal women. Figure 1 The change of blood PCSK9 and lipid pro les in ovariectomized mice and their correlation analysis A. PCSK9 in the blood increased in ovariectomized mice, B1-B4. Lipid pro les in the blood; C1-C2. The correlation analysis between PCSK9 and TC under the two diets; D1-D2. The correlation analysis between PCSK9 and LDL-c under the two diets; * represents p<0.05, ** represents p<0.01, *** represents p<0.001 The change of PCSK9 and LDLR in the liver of ovariectomized mice A. The expression of PCSK9 and LDLR in the liver of ovariectomized mice by IHC, B1-B3: The expression of PCSK9 and LDLR in the liver of ovariectomized mice with normal diet by WB; C1-C3: The expression of PCSK9 and LDLR in the liver of ovariectomized mice with high-fat diet by WB; * represents p<0.05, ** represents p<0.01 PCSK9 inhibitor improved the lipid disorders in ovariectomized mice A. PCSK9 in the blood of the mice; B1-B2.TC and LDL-c in the blood of the mice. C: IHC examined the expression of PCSK9 and LDLR in the liver of the ovariectomized mice treated with estrogen, PCSK9 alone or both together; D1-D3: WB examined the expression of PCSK9 and LDLR in the liver of the ovariectomized mice treated with estrogen, PCSK9 alone or both together * represents p<0.05, ** represents p<0.01