Inhibition of Neuropilin-1 Improves Non-Alcoholic Fatty Liver Disease via PI3K/AKT/Mtor Signaling in High-Fat-Diet induced Obese Mouse

Background: Research ndings indicate Neuropilin-1 plays a critical role in lipid metabolism and obesity-associated insulin resistance, on such a basis, this study aims to explore the effects and working mechanism of Neuropilin-1 inhibition on the non-alcoholic fatty liver disease in high-fat-diet induced obese mice. Methods: Firstly, the pcDNA3.1-NRP-1 recombinant plasmid containing Neuropilin-1 gene and the Neuropilin-1 gene RNA interference plasmid shRNA-NRP1 were successfully constructed. A total of 36 C57BL/6 mice were randomly assigned to 6 groups, blank group, control group, pcDNA3.1 injection group, pcDNA3.1-NRP-1 injection group, pGenesil-1.1 injection group and shRNA-NRP1 injection group. Expression of phospho-PI3K, phospho-AKT, phospho-mTOR and Neuropilin-1 in liver was measured as well as body and liver weight, blood glucose, serum transaminases and lipid levels of the mice. Results: The weight and liver mass of high-fat-diet fed mice injected with pcDNA3.1-NRP-1 were signicantly higher than those from the control group, but their body weight and liver mass decreased signicantly after shRNA-NRP1 injection. The results also showed that Neuropilin-1 expression can signicantly inuence the severity of hepatic steatosis in high-fat-diet fed mice, decreased serum FPG, LDL, AST, ALT levels and the expression of TNF-α, IL-1β, and IL-6 mRNA. In addition, the Neuropilin-1 expression will also inuence the p-PI3K, p-AKT and p-mTOR in mice. Conclusions: This study concluded that the inhibition of Neuropilin-1 could improve Non-alcoholic fatty liver disease by decreasing body weight and reduce inammation in high-fat-diet induced obese mice by modulating the PI3K/AKT/mTOR signaling pathway. multiple cloning sites (MCS) of the plasmid pGenesil-1.1 were as follows: MluI-hU6promoter- InsertDNA-SacI, a SacI cleavage site was designed in the fragment of inserted target gene, and the plasmid pGenesil-1.1 itself had a SacI. The cleavage site, if inserted correctly, the plasmid can be cleaved by SacI to a 916 bp DNA band. Enzyme digestion was performed to identify the correct plasmid sequencing. HFD was reduced by the regulation of NRP1 expression, but elevated through an enhance NRP1 expression. HFD leads to inammation and weight gain, and the inhibition of NRP1 expression could alleviate the inammation as well as obesity caused by HFD. Taken together, our data indicate that NRP1 affects the PI3K/AKT/mTOR signaling pathways leading to elevated inammatory responses induced by HFD.

capture by cell membrane, thereby enhancing TGF-β1 signal transduction and promoting TGF-β1 secretion.
Furthermore, promoted the epithelial-mesenchymal transition (EMT) [20,21]. Recently, Dai et al. have revealed the new and crucial role that myeloid cell NRP1 might have played during the modulation of the insulin resistance associated with obesity [22]. In another research, Zhang et al. found that even though the mice were fed with a highfat diet, those lacked the genes encoding NRP1 and VEGFR1 did not consume lipids in a normal fat speed, and they excreted lipids instead, therefore, the mice rarely gained weight [23].
These research ndings thus con rmed the critical role that NRP1 plays in lipid metabolism and insulin resistance that associated with obesity, however, there's still no report covering the correlation between NRP1 and HFD-induced NAFLD. PI3K/Akt/mTOR signaling pathway plays an important role in the occurrence and development of liver brosis [24]. Activated PI3K/Akt/mTOR promoted the formation and development of liver brosis by regulating the proliferation and migration of hepatic stellate cells (HSC), increasing the expression of Collagen and affecting the balance of MMP and TIMP [25]. Therefore, blocking PI3K/Akt/mTOR signaling pathway can induce the apoptosis of HSC, inhibit the secretion of ECM and promote the degradation of ECM, thus delaying the progress of liver brosis.
Both NRP-1 and PI3K/Akt/mTOR signal transduction pathways have effects on hepatic stellate cells (HSC), and further promote the occurrence of liver brosis. However, there has been no research on the correlation between NRP-1 and PI3K/Akt/mTOR signal transduction pathways. However, there is no study showed the correlations between NRP-1 and PI3K/Akt/mTOR signal transduction pathways, thus, this study was designed to investigate the effect of NRP1 on HFD-induced NAFLD and to explore the underlying mechanism via PI3K/AKT/mTOR signaling.

Materials And Methods
Bacteria, carriers and experimental reagents pGenesil-1.1, pCR-blunt, pcDNA3.1, DH5α strains were from the laboratory of research center; SYBR Premix EX Taq, T4 DNA ligase, Taq DNA polymerase were purchased from TaKaRa; EcolI, Hind III, SacI endonucleases were purchased from NEB Company; RNeasy kit, plasmid extraction kit and gel recovery kit were purchased from GIAGEN; Trizol, liposome Lipofectamine TM2000 transfection kit was purchased from Invitrogen; P38, p-P38, JNK, p-JNK, NRP-1 antibodies were purchased from Cell Signaling Technology; NRP1 interference sequence synthesis, primer synthesis and gene sequencing were performed by Shanghai Shenggong Bioengineering Technology Service Co.,

Ltd.
Construction and identi cation of recombinant plasmid pcDNA3.1-NRP-1 Construction of pCR-blunt-NRP-1 cloning vector Total RNA of mouse liver tissue was extracted by TRIzol method, NRP-1 gene was ampli ed by reverse transcription, PCR product was subjected to agarose gel electrophoresis, and the target gene was recovered. Fragments of the target gene were ligated with the pCR-blunt vector by T4 DNA ligase, and then the product transformed competent cells, the monoclonal colony was picked overnight. The small amount of the extracted plasmid was identi ed by EcolI single digestion and sequenced. After sequencing, the plasmid was correctly sequenced and named pCR-blunt-NRP-1.
Construction and identi cation of pcDNA3.1-NRP-1 expression vector The recombinant plasmids pCR-blunt-NRP-1 and pcDNA3.1 were digested with EcolI, and the reaction product was detected by 0.7% agarose gel electrophoresis and the target band was observed. The GRP-recovered NRP-1 gene fragment was ligated with pcDNA3.1, T4DNA ligase. The ligation product then transformed competent cell DH-5α.
On the second day, several monoclonal colonies were picked, of which the plasmid was extracted after shaking the bacteria and placed its overnight, and the recombinant plasmid was identi ed by single digestion with Hind III. The correctly digested plasmid was named pcDNA3.1-NRP-1, and the bacterial solution was stored at -80°C.
Construction of NRP1 gene RNA interference plasmid of shRNA-NRP1 An interference sequence of 19 nt was designed based on the NRP1 gene mRNA sequence: NRP1: GGACCCATACCAGAGAATT (target 706 bp); NRP-1A: 5'-CACCGGACCCATACCAGAGAATTCTATGGACAAATTCTCTGGTATGGGT CCTTTTTTG-3', NRP-1B: 5'-AGCTCAAAAAAGGACCCATACCAGAGAATTT GTCCATAGAATTCTCTGGTATGGGTCC-3', primer structure: CACC + Sense + Loop + Antisense + termination signal + SacI. The sense strand was anneal-linked to the single strand of the antisense oligonucleotide, and the double-stranded oligonucleotide was ligated to the pGenesil-1.1 linear vector (containing the hU6 promoter) by T4 DNA ligase, and the ligated product was transformed into E. coli DH5α, and inoculated the incubated overnight at 37°C on kanamycin resistant LB plates. Monoclonal colonies were picked, inoculated into kanamycin-resistant LB medium, and cultured overnight at 37°C in a constant temperature shaker. The plasmid was extracted and identi ed by SacI digestion. The multiple cloning sites (MCS) of the plasmid pGenesil-1.1 were as follows: MluI-hU6promoter-InsertDNA-SacI, a SacI cleavage site was designed in the fragment of inserted target gene, and the plasmid pGenesil-1.1 itself had a SacI. The cleavage site, if inserted correctly, the plasmid can be cleaved by SacI to a 916 bp DNA band. Enzyme digestion was performed to identify the correct plasmid sequencing.

Experimental design
A total of 36 male C57BL/6 mice (6 weeks old, 18.44 ± 0.92 g) were purchased from experimental animal center of Tongji Medical College a liated to Huazhong University of Science and Technology. The mice were housed in a clean room maintained at 24 ± 2°C in a 12h: 12h light: dark cycle, with free access to food and water. All animal experiments were approved by the Animal Care and Use Committee of Tianyou Hospital a liated to Wuhan University of Science and Technology with the Ethics Protocol number TY20180212. Mice were randomly assigned to 6 groups. Group A (n = 6) were fed with regular diet (RD) for 16 weeks, and the other groups were fed with HFD for 16 weeks. After 8 weeks, Group B (n = 6) received 100 ug/kg physiological saline by intravenous injection, Group C (n = 6) received 100 ug/kg pcDNA3.1 by intravenous injection, Group D (n = 6) received 100 ug/kg pcDNA3.1-NRP-1 by intravenous injection, Group E (n = 6) received 100 ug/kg pGenesil-1.1 by intravenous injection, Group F (n = 6) received 100 ug/kg shRNA-NRP1 by intravenous injection. All the groups received 2 injections in a week (on Monday and Friday respectively) ( Table 1). HFD and RD came from Meidisen Biomedical Company (Jiangsu, China). The diets for mice in the experiment were in accordance with AIN93 recommendations, of which 60% calories of the high-fat diet (research diet D12492) came from fat, 20% from proteins and the rest 20% from carbohydrates. Of the regular diet (research diet D12450B), 10% calories came from fat, 20% from proteins and 70% from carbohydrates.
In the end of the experiment, mice were anaesthetized with pentobarbital sodium, livers were removed and blood was collected via cardiac puncture.

Evaluation of body and liver weight
Body weight of the mice was measured every 2 weeks consistently between 7 pm and 8 pm, liver weight was measured at the end of the experiment after sacri ce.

Levels of blood transaminase and lipid
The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), triglycerides (TG), lowdensity cholesterol (LDL-C) and high-density cholesterol (HDL-C) were measured spectrophotometrically in accordance with the instructions (NJJCBIO, Jiangsu, China).

Histological analyses
Took a representative mouse from each group, then took the liver and epididymal adipose tissue of the mice and xed them in 10% buffered formalin, embedded in para n, cut into thick sections of 8 µm. Stained with hematoxylin and eosin for histological examination of lipid droplets, and then took an Olympus SZX10 microscope (Tokyo, Japan) to get the images. The software Image Pro Plus 7.01 (Media Cybernetics, Silver Springs, MD, USA) was taken to analyze the volume density (Vv) of hepatic steatosis, and the results are listed as below [26].

Real-time reverse-transcriptase polymerase chain reaction (qRT-PCR)
Trizol was taken to isolate the total mRNA of mouse liver. According to the instruction, two micrograms of total RNA were used for cDNA synthesis with the help of the RNase kit. 7900HT real-time PCR system (ABI, CA, USA) was taken to perform a quantitative real-time SYBR Green quantitative RT-PCR, and the expression of target genes was determined. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA was taken as an endogenous control and the ΔΔCt method was used for the analysis of relevant data. Primer sequences are provided in Table 2.

Statistical evaluation
Statistical analyses were performed using software of SPSS 21.0 (Chicago, IL) and GraphPad Prism (GraphPad Software Inc). Data were presented as mean values ± standard errors. The one-way ANOVA test was used to determine differences between groups by comparing the internal variability of the control groups with the variability among all experimental groups. Multiple comparisons between the groups were performed using Student-Newman-Keuls method as a post hoc test. P < 0.05 was considered statistically signi cant. All assays were performed in triplicate.

NRP1 mRNA
As shown in Fig. 1, the expression of NRP1 mRNA in liver tissue of group D was signi cantly higher than that of other groups (P < 0.05). The expression of NRP1 mRNA in liver tissues of mice in group A, B, C and E was signi cantly higher than that in group F (P < 0.05), but there was no signi cant difference in the expression of NRP1 mRNA in liver tissues of mice in group A, B, C and E (P > 0.05).

NRP1 protein
As shown in Fig. 2A and Fig. 2B, the expression of NRP1 protein in liver tissue of mouse from group D was signi cantly higher than that of mouse from other groups (P < 0.05). The expression of NRP1 protein in liver tissues of mouse in group A, B, C and E was signi cantly higher than that of mouse from group F. There was no signi cant difference between the expression of NRP1 protein in liver tissues of mouse from group A, B, C and E (P > 0.05).

Body weight and liver mass
As shown in Fig. 3A, at week 8, the weight of mouse in group A was signi cantly lower than that in other groups (P < 0.05). The weight of mouse in group D increased signi cantly after a tail injection of pcDNA3.1-NRP-1, and their body weight was signi cantly higher than that of the mouse from Groups B, C, and E at all time points (P < 0.05).
The body weight of mouse from Group F decreased signi cantly after shRNA-NRP1 injection in the tail vein, and the body weight of mouse in this group was signi cantly lower than that of mouse from Group B, C, E at each time point (P < 0.05), but the body weight of mouse from Group B, C, and E groups was not signi cantly different at any time point (P > 0.05). As shown in Fig. 3B, the liver mass of mouse from Group D was signi cantly higher than that of mouse from other groups (P < 0.05), and the liver mass of mouse from Group B, C and E was not signi cantly different (P > 0.05), but was signi cantly higher than that of mouse from Group A and F (P < 0.05), the liver mass of mouse from Group A and F was not statistically signi cant (P > 0.05).

Glucose and lipid metabolism
As shown in Table 3, the serum levels of FPG, LDL, AST and ALT of mouse in group D were signi cantly higher than those of mouse in other groups (P < 0.05). The serum levels of FPG, LDL and AST of mouse in group B, C and E were similar. The levels of ALT and ALT of mouse from groups B, C, D, E were not signi cantly different (P > 0.05), but they were signi cantly higher than those of mouse in group A and F (P < 0.05). There was no statistical difference in serum FPG, LDL, AST and ALT levels between mouse in Group A and Group F (P > 0.05).

Histological analyses of liver
As shown in Fig. 4A-D, the liver steatosis ratio and adipocyte diameter of mouse in Group D were signi cantly higher than those of mouse in other groups (P < 0.05). There was no signi cant difference in liver steatosis ratio and adipocyte diameter between mouse in Group B, C and E. (P > 0.05), but the levels were signi cantly higher than those of mouse in Group A and F (P < 0.05), and there was no signi cant difference in liver steatosis ratio and adipocyte diameter between mouse in Group A and F (P > 0.05).
Quanti cation of pro-in ammatory cytokines As shown in Fig. 5, the mRNA levels of TNF-α, IL-1β and IL-6 in the liver tissues of mouse in Group D were signi cantly higher than those of mouse in other groups (P < 0.05). The levels of TNF-α, IL-1β and IL-6 mRNA of liver tissue of mouse in Group B, C and E was no signi cantly different (P > 0.05), but they were signi cantly higher than those of mouse in Group A and F (P < 0.05). The levels of TNF-α, IL-1β and IL-6 mRNA of liver tissue of mouse in Group A and Group F were not signi cantly different (P > 0.05), and the levels of IL-10, JNK and IKK-β mRNA of liver tissue of mouse in all groups were not signi cantly different (P > 0.05).
The related proteins expression of PI3K/AKT/mTOR signaling pathway As shown in Fig. 6A and Fig. 6B, There were no signi cant differences in PI3K, AKT, mTOR protein levels between the liver tissues of mouse in all groups(P > 0.05).There were no signi cant differences in p-PI3K,p-AKT, p-mTOR protein levels between the liver tissues of mouse in Group B, C, E(P > 0.05), but they were signi cantly higher than those of mouse in Group A and F(P < 0.05), and lower than those of mouse in Group D(P < 0.05).There were no signi cant differences in p-PI3K,p-AKT, p-mTOR protein levels between the liver tissues of mouse in Group A and F(P > 0.05). The phosphor/total ratio of each protein were shown in Fig. 6C.

Discussion
NRP-1 is an important member of the NRP family and plays an important role in neurodevelopment, angiogenesis, tumor invasion, metastasis and immunity [28][29][30][31]. NRP-1 initially has been identi ed as a coreceptor for Sema3 and growth factors, including vascular endothelial growth factor, transforming growth factor-b, hepatocyte growth factor and platelet-derived growth factor [32][33][34][35], and it have been shown to be strongly expressed in immune cells and to regulate immune response [36,37]. In macrophages, ablation of NRP1 results in decreased tumor growth and metastasis via enhanced in ltration of tumor-associated macrophages into normoxic tumor regions, which abolishes the proangiogenic and immunosuppressive functions of tumor-associated macrophages [38]. Consistently, Miyauchi et al. reported that NRP1 ablation in glioma-associated microglia and macrophages suppresses glioma progression by promoting M1 macrophage polarization [39]. Recent ndings further established the involvement of the Sema3A/Nrp1 axis, as well as M1/M2 macrophages, with respect to tumorigenic processes [40]. However, speci c functions of NRP1 in the context of metabolic dysfunction, such as NAFLD, have not been investigated yet. Studies have shown that: NRP1 may play an important role in the process of capturing TGF-β1 and LAP-TGF-β1 by cell membrane, thereby enhancing TGF-β1 signal transduction and promoting TGF-β1 secretion, thus promoting the occurrence and development of EMT [21]. EMT-producing cells may enhance cell hardness and provide brotic cytokines. Indirect stimulation of myo broblasts induces brosis [41]. Other studies have shown that NRP-1 plays a novel role in tumor progression by enhancing the autorine HGF/ c-Met pathway, suggesting that NRP-1 may also play a role as a functional receptor for HGF [42,43]. HGF regulates the survival, proliferation and migration of endothelial cells, matrix deposition and degradation, and the formation of capillary-like structures through c-Met [44]. All the above studies suggest that NRP-1 can in uence brosis, but its role in HE has not been reported in the literature.
This study is to observe the effect of NRP-1 expression enhancement and inhibition of NRP-1 expression on the formation of HFD-induced obese mouse NAFLD, and to explore its mechanism of action. Long-term high fat diet feeding in mice could cause whole-body energy imbalance, resulting in a series of obesity-associated metabolic disorders. To study the relation about NRP-1 and NAFLD, we established a NAFLD model. The results suggested that elevated NRP-1 expression can signi cantly increase the body weight and liver mass of NAFLD model mouse, while inhibition of NRP-1 expression can signi cantly reduce the body weight and liver mass of NAFLD model mouse.
The results also suggest an increase in NRP-1 expression can aggravate the abnormality of blood lipid and liver function of NAFLD model mice, and inhibiting the expression of NRP-1 can signi cantly alleviate the abnormality of blood lipid and liver function of NAFLD model mouse. In addition, H&E staining results also indicated that the increase of NRP-1 expression may aggravate the degree of liver degeneration of NAFLD model mouse and inhibit the expression of NRP-1. The degree of NAFLD model mouse liver degeneration can be signi cantly reduced.
Insulin resistance is considered to be the central link in the development of NAFLD and metabolic syndrome. In ammatory factors affect insulin sensitivity, and in ammatory factors are involved in the development and progression of NAFLD, adipose tissue and liver immune dysfunction in patients with NAFLD, abnormal macrophages. Macrophages cells are important sources of in ammatory factors, and both fat and hepatocytes secrete in ammatory factors such as TNF-α and IL helps to mediate macrophage in ltration [45,46]. To explore the possible mechanism of NRP1's effects on NAFLD, we measured mRNA levels of pro-in ammatory makers. Then we found that the inhibition of NRP1 expression signi cantly reduced the mRNA level of pro-in ammatory cytokines including TNF-α, IL-1β, and IL-6, which were increased in HFD fed mouse, but the enhanced NRP1 expression signi cantly elevated the mRNA level of pro-in ammatory cytokines.
NAFLD is currently considered to be a metabolic stress liver injury closely related to insulin resistance and genetic susceptibility, in which insulin resistance; oxidative stress and in ammatory response play an important role in the development of NAFLD [47][48][49]. As a key pathway of insulin signaling, PI3K/Akt/mTOR participates in glycolipid metabolism by regulating insulin level [50]. However, PI3K/Akt/mTOR signal transduction disorders caused by various factors can cause insulin resistance (IR), promote the occurrence and development of NAFLD [51], and also participate in the pathophysiological process of oxidative damage of cells [52]. Phosphatidylinositol 3kinase/protein kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) was an important signal transduction pathway in cells, which was closely related to cell proliferation, apoptosis, angiogenesis and other biological behaviors [53]. PI3K was an important member of the superfamily of growth factor receptor signal transduction process. AKT and mTOR were important downstream genes of PI3K, and PI3K could affect families related to cell apoptosis through AKT, mTOR and effector molecules, thereby affecting cell apoptosis [54]. In ammation were a major factor in the occurrence and development of chronic liver disease and lead to liver brosis and cirrhosis, PI3K and AKT could adjust the key in ammatory cytokine activation of immune cells [55], PI3K and mTOR limited the activation of immune cells, adjusted in the liver in ammation and brosis formation, regulated the proliferation and migration of HSC in the process of liver brosis, synthesis and degradation of the ECM by raising the key antiin ammatory cytokine interleukin-10 and inhibiting proin ammatory cytokines [56]. In addition, in the state of in ammation, the PI3K/AKT/mTOR signaling pathway was over-activated, resulted in excessive transcription of downstream target genes, abnormal cell proliferation, tissue repair disorders, and aggravating tissue damage.
Based on the consensus that "IR→abnormal lipid metabolism→liver fat deposition →oxygen stress and lipid peroxidation→NAFLD changes", the PI3K/AKT/mTOR signaling pathway was expected to be the target for improving NAFLD. To explore the possible mechanism of NRP1's effects on NAFLD, we measured the active levels of PI3K/AKT/mTOR signaling. The expression of PI3K, AKT and mTOR were similar in six groups, but the phosphorylated levels of PI3K, AKT and mTOR increased in HFD fed mouse when compared with that of RD fed mouse. The activation of the PI3K/AKT/mTOR signaling molecules by HFD was reduced by the regulation of NRP1 expression, but elevated through an enhance NRP1 expression. HFD leads to in ammation and weight gain, and the inhibition of NRP1 expression could alleviate the in ammation as well as obesity caused by HFD. Taken together, our data indicate that NRP1 affects the PI3K/AKT/mTOR signaling pathways leading to elevated in ammatory responses induced by HFD.

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There are still some limitations in this study. Only the effects of enhancement or inhibition of NRP1 expression on proteins related to PI3K/Akt/mTOR signal transduction pathway were studied, and it was concluded that NRP1 may have an effect on non-alcoholic fatty liver disease by regulating PI3K/Akt/mTOR signal transduction pathway.
Further investigation is needed through the use of PI3K/Akt/mTOR signaling pathway agonists or inhibitors for interference. In addition, does NRP1 regulate other signaling pathways besides PI3K/Akt/mTOR signaling pathway to exert in uence on non-alcoholic fatty liver disease? This will require further study.

Conclusions
This study concluded that HFD leads to in ammation and weight gain, and the inhibition of NRP1 expression could alleviate the in ammation as well as obesity caused by HFD. Taken together, our data indicate that NRP1 affects the PI3K/AKT/mTOR signaling pathways leading to elevated in ammatory responses induced by HFD.

Declarations
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.    Figure 1 The relative expression of liver NRP-1 mRNA at every group (n = 6). Statistical differences between groups with various treatments were identi ed using one-way ANOVA followed by the Student-Newman-Keuls (SNK) test; # P<0.05 for group D compared with other groups; *P<0.05 for group A, B, C, E compared with group F.   groups with various treatments were identi ed using one-way ANOVA followed by the Student-Newman-Keuls (SNK) test; the adipocyte diameter of mice at every groups (n = 6). #P<0.05 for Group D compared with other groups; *P<0.05 for Group B, C, E compared with Group A, F.