Protective effects of PAMAM dendrimers against ANIT-induced cholestatic liver injury in mice CURRENT STATUS: REVIEW

Background:Cholestatic liver disease (CLD) is a common disease of infancy, threatening infants’ health seriously. However, there is no effective drug to treat this disease.It is urgently to develop a new drug to overcome it. Polyamide-amine (PAMAM) dendrimer is a hyperbranched nano polymer, which has been found that has an anti-inflammatory effect recently. For this study, we aim to explore the protective effect and mechanism of PAMAM on CLD. Methods:In this study, a mouse model of cholestatic liver injury was created using ANIT,and TNF-α was utilized to stimulate human hepatocytes to investigate the protective effect and mechanism of 4.5th generation carboxyl-terminated PAMAM dendrimers (G4.5-COOH) in vivo and in vitro.Liver serum biochemistry, inflammatory, oxidative stress(OxS), endoplasmic reticulum(ER) stress and apoptosis indicators were determined.In addition, the eﬀects of G4.5-COOH on PPAR-γ and PI3K/AKT/mTOR signaling pathway were studied. Data were analyzed by using one-way ANOVA. Results:We find the G4.5-COOH may inhibit TNF-α to damage hepatocytes and ameliorate the cholestatic liver injury. We also find that the protective effect of G4.5-COOH might be due to inhibition of P-AktP-mTOR expression by a mechanism that might be partly dependent on up-regulated functional expression of PPAR-γ. Conclusions:G4.5-COOH PAMAM dendrimer has a protective effect on CLD.

3 cause liver cirrhosis. [3]ANIT is a toxic reagent for the bile duct. Single-dose oral administration can induce acute biliary inflammation, leading to bile duct obstruction and producing intrahepatic cholestasis, which is similar to the pathological characteristics of human CLD and rat bile duct ligation model. [4]Therefore, it is an ideal model for the study on CLD.
PAMAM dendrimers are hyperbranched nanopolymers with diamine (usually ethylenediamine) as the core radiating from the center. They have well-defined controllable spherical structure and nanoscale. [5]Their internal cavity and high-density surface groups can increase the solubility and bioavailability of drugs through physical encapsulation, electrostatic interaction and covalent binding of drugs, genes, etc. They have been widely used in biomedical fields such as drug delivery, gene therapy, medical imaging and diagnosis, which are one of the most promising nanocarriers. [6][7][8]Recent studies have found that PAMAM itself has some biological activities and functions. For example, Chauhan et al. unexpectedly discovered that unmodified naked PAMAM with simple surface groups (such as -NH 2 , -OH, etc.) had unexpected anti-inflammatory properties. [9]Yin et al. revealed that anionic and neutral PAMAM dendrimers had protective effects on acute pancreatitis in mice, and their anti-inflammatory mechanism was related to the inhibition of nuclear translocation of NF-κB in macrophages [10].
Cholestatic liver injury induces the accumulation of inflammatory cells and the secretion of proinflammatory cytokines. It has been proved that cholestatic liver injury is related to the re-emergence of inflammatory factors such as TNF-α, IL-6 and IL-1β. [11,12] Inflammatory cytokines are essential for inflammation, hepatocyte death, subsequent regeneration and hepatic fibrosis during the process of cholestatic liver injury. [3] Bile acids have been reported to contribute to the synthesis and excretion of IL-1β and TNF-α. [13]PAMAM has an anti-inflammatory effect. It has been confirmed that G4.5-COOH has a 4 certain therapeutic effect on a mouse model of pancreatitis. What about its effect on the liver? At present, the effect of PAMAM on the liver has not been reported. Therefore, we consider whether PAMAM can be applied to the prevention and treatment of liver diseases.

Synthesis of G4.5-COOH PAMAM Dendrimers
The G4.5-COOH was synthesized as described earlier with minor modification [14,15]. The G4.5-COOH was synthesized as follows: The PAMAM dendrimers were synthesized by two steps at 25 °C according to a classical method. After vacuum rotation evaporation and G4.5-COOCH 3 were eventually obtained. In order to prepare G4.5-COOH, a certain volume of G4.5 PAMAM aqueous solution was placed in a round bottom flask, then 0.2M HCl solution was added dropwise to adjust the pH of solution to 1-2 and react at room temperature for 72 h. The synthesized dendrimers of G4.5-COOH were purified by membrane dialysis to remove a portion of the trailing generation defect structures.

Mice and Treatments
Healthy adult male Balb/C mice were supplied by the Experimental Animal Center of Hebei Medical University (Certificate no.:1708105,China). Mice were housed in a hygienic environment with a constant room temperature, and they had free access to food and water. All animals were acclimatized for 2 weeks prior to experiment.
Mice were divided into four groups with six mice per group. Mice in control group was treated intravenously (i.v.) with normal saline (NS) q.o.d. for 2 weeks,and on the twelfth day were given the vehicle(olive oil) alone. ANIT group was treated with normal saline (NS) q.o.d. for 2 weeks and severally administrated with 100 mg/kg ANIT(TCI,Japan) (dissolved in olive oil)on the twelfth day.-COOH + ANIT group was treated intravenously (i.v.) with G4.5-COOH at a dose of 50 mg/kg q.o.d. for 14 days according to a regimen published by literature [10] and severally administrated with 100 mg/kg ANIT(dissolved in olive oil)on the twelfth day; GW9662+-COOH + ANIT group was treated with GW9662(ApexBio,USA)3 mg/kg [16] at 0.5 h before G4.5-COOH reatment,the rest of steps are the same as -COOH + ANIT group. The assay method of SOD ,GSH and MDA levels in liver tissues and THLE cells was following the instructions of the kits provided by Suzhou Geruisi Biotechnology Co., Ltd (China).

Gene Expression Assay
Total RNA from liver tissues was extracted by utilizing TRIzol reagent(TIANGEN Biotech,Beijing), and first-strand cDNA was generated by a RT kit (TaKaRa Bio,Japan).

Flow Cytometry
The percentage of apoptotic THLE cells was measured using a commercial kit (Absin, 7 China). THLE cells were stained with FITC-labelled Annexin V (1 mg/mL) and propidium iodide (10 mg/mL) for 15 min. After gentle washes, patterns of apoptotic cells were measured using a FACSVerse (Becton-Dickinson, USA).

Western Blots
Tissues and cells were lysed with RIPA buffer (Solarbio,China) respectively. The concentration of total lysate was measured by using a BCA kit (Solarbio,China). A total of 30ug samples were separated on a 10% SDS-PAGE gel and transferred onto 0.4 mM PVDF membranes(MILLPORE,USA). The membranes were blocked with 5% BSA-PBST buffer for 1 h, followed by the incubation with primary anti-bodies overnight at 4℃ and HRP-linked secondary antibodies (KPL,USA) for 2 h at RT. Bands were detected using enhanced chemiluminescence (ECL) prime reagent (Vazyme,China). Intensity of bands was assessed using Image-J software.

Statistics
The results are expressed as mean ± SD. The statistical significance of the experimental data was analysed by one-way ANOVA. P < 0.05 was considered statistically significant.

Characterization of G4.5-COOH PAMAM Dendrimers
The FTIR spectrum of G4.5-COOCH3 and G4.5-COOH was shown in After gavage administration with ANIT, the mice presented mental sluggishness, messy and dull hair, dementia and physical inactivity, decreased appetite, reduced drinking, and dark yellow urine opposed to the normal group. The livers were dark red with white necrotic foci, the gallbladders were significantly enlarged with cholestasis, and the serum was dark and turbid. Report to ANIT group, -COOH + ANIT group showed significant improvement in spirit, hair, appetite and activity, the color of urine and liver was obviously lighter, the gallbladders were smaller and serum was clearer. However, after adding GW9662, the mice showed poorer spirit, hair, appetite and activity, darker urine, darker livers, larger gallbladders and more turbid serum than those in -COOH + ANIT group ( Fig. 2).

G4.5-COOH Reduced Inflammation induced by ANIT treatment in Mice
The control group showed a normal portal triad comprising of the bile duct, hepatic artery and portal vein. In ANIT group, dilatation and hemorrhage of the portal vein, bile duct epithelial hyperplasia, bile duct stenosis and cholestasis were observed. Moreover, a large number of inflammatory cells infiltrated the portal area, with punctate and focal necrotic areas. In -COOH + ANIT group, the bile duct epithelium was slightly damaged and inflammatory cell infiltration was rare, almost returned to the conditions of the control group. In GW9662 + -COOH + ANIT group, bile duct epithelial hyperplasia, inflammatory 9 cell infiltration and focal necrosis of the liver were increased (Fig. 4.A).

G4.5-COOH Decreased Cell Viability in A Concentration-Dependent Manner
In order to evaluate the cytotoxicity of G4.5-COOH and select appropriate concentration for further analysis, CCK8 was carried out. The results showed that G4.5-COOH decreased cell viability in a concentration-dependent manner. We chose the concentration of 7 uM which had little effect on cell viability for following experiments (Fig. 5).

G4.5-COOH Reduced OxS in Cholestatic Liver Injury
Compared with the control group, SOD and GSH levels in liver tissues of ANIT group were significantly lower ( P 0. 001) while MDA level was significantly higher( P 0. 001); Compared with ANIT group, the levels of SOD and GSH in -COOH + ANIT group increased significantly( P 0. 001), while the level of MDA decreased significantly( P 0. 001).

G4.5-COOH Reduced Liver Apoptosis in Cholestatic Liver Injury
The TUNEL staining and Bax Bcl-2 protein results showed that compared with control group, the apoptosis rate was increased in ANIT group and reduced in -COOH + ANIT group(P < 0.05), indicating that G4.5-COOH inhibited liver cell apoptosis. However, after adding GW9662, the apoptosis rate was raised again(P < 0.05),indicating that GW9662 can antagonize the action of G4.5-COOH (Fig. 8.A-C).
Furthermore, THLE cells were incubated with Annexin V and propidium iodide (PI)dye and detected by a flow cytometer. The Flow Cytometry and Bax Bcl-2 protein results showed that the apoptosis rate was significantly increased after interfering with TNF-α(P < 0.05), while G4.5-COOH reduced the rate of apoptosis and GW9662 increased the rate of apoptosis again P < 0.05 .These results indicated that G4.5-COOH could inhibit THLE cell apoptosis which induced by TNF-α (Fig. 8.D-F).

G4.5-COOH increased PPAR-γ expression through inhibiting PI3K/ Akt/mTOR signaling pathway in Cholestatic Liver Injury
Next, we ought to investigate whether PI3K/Akt/mTOR signaling pathway was involved in the increase PPAR-γ induced by G4.5-COOH.The protein levels of PPAR-γ was downregulated and p-Akt, p-mTOR were unregulated when exposing to ANIT or TNF-α(P < 0.05), and G4.5-COOH treatment further unregulated PPAR-γ expression and down-regulated p-Akt, p-mTOR expressions compared with ANIT or TNF-α group(P < 0.05), while the protein levels of Akt and mTOR were not significantly different between groups(P > 0.05). To further clarify the result, PPAR-γ inhibitor GW9662 was administrated to the mice and cells. Compared with G4.5-COOH + ANIT group, GW9662 markedly decreased the protein levels of PPAR-γ P < 0.05 ,demonstrating that the inhibitor was effective. However at the same time the phosphorylation of mTOR and Akt was up-regulated again P < 0.05 . (Fig. 9.A-D)

Discussion
The molecular mechanism underlying the early liver injury associated with cholestasis remains unclear. It is generally accepted that high-concentration bile acids in hepatocytes and blood caused by cholestasis can produce direct toxicity to hepatocytes and induce hepatocyte apoptosis [19,20]. However, with the development of analysis and detection technology, the quantitative determination of various bile acid components in the vivo model of cholestasis demonstrated that the concentration of bile acids was obviously insufficient to reach the concentration level that could directly cause cytotoxicity in vitro experiments, indicating that cholestatic liver injury has other pathogenesis besides the toxicity of bile acids [21][22][23]. Therefore, researchers began to focus on the role of inflammation and immune response caused by cholestasis on liver injury [24,25]. The literature reported that 24 h after bile duct ligation, a large number of inflammatory cells infiltrated around the injured liver tissue [26,27]. Our study also showed the necrosis of diseased hepatocytes accompanied by inflammatory cell infiltration in mice 48 h after ANIT administration. When the liver is stimulated by external trauma or harmful substances, macrophages are firstly activated to play a role of immune defense for phagocytosis and remove the harmful substances to protect the liver. However, with the secretion of inflammatory factors, excessive or unsolvable inflammatory reaction is nearly always accompanied by a massive loss of hepatocytes, resulting in irreversible damage to the hepatic parenchyma [28,29]. TNF-α is part of the most important inflammatory factors.
Numerous studies have shown that TNF-α is closely linked to liver diseases. It is not only an essential mediator of the inflammatory reaction, but also directly damages hepatic parenchymal cells [30,31]. Our results confirmed extensive macrophage infiltration around the bile ducts, vessels and necrotic areas,and significantly increased TNF-α secretion in mice with cholestasis. Further, it showed that G4.5-COOH effectively inhibited the above inflammatory response.
Recent studies have found that ER stress plays a major role in various liver diseases, and its mechanism may be related to ER stress-induced apoptosis, which is a new hotspot in medical research [32,33]. ER stress plays an important role in the body's inflammatory response. The effects of ER stress and inflammation are not unilateral. Inflammatory factors can also activate ER stress. TNF-α, IL-1β and IL-6 can induce ER stress in hepatocytes and activity the cAMP response element binding protein H(CREBH),thus mediate an acute-phase response(ARP) [34],The mechanism may be related to inflammatory factors promoting calcium release and ROS aggregation in the ER, thus interfering with the correct folding of proteins and mitochondrial metabolic balance [35].
Xue X et al. reported that TNF-α induced ROS accumulation and cell death. TNF-α induced unfolded protein response (UPR) in a ROS-dependent manner, resulting in ER stress [36].This takes into account the relationship among inflammation, OxS and ER stress.Our study is consistent with the above findings that TNF-α induced ER stress in hepatocytes, and ANIT-induced cholestasis not only caused inflammation and OxS, but also caused ER stress injury in the mice liver. We are equally surprised to discover that G4.5-COOH has some effect on inhibiting ER stress.
Under normal physiological homeostasis, there is a dynamic balance between the number of newborn cells and apoptosis cells. Once this balance is broken (a relative increase in proliferating cells or apoptosis cells), pathological damage will be induced. Inflammation, OxS and ER stress can result in apoptosis [37,38]. Our study revealed that the liver of mice with cholestasis induced by ANIT gavage had extensive apoptosis and necrosis. In vitro studies also found that TNF-α could induce the apoptosis of hepatocytes, while G4.5-

COOH effectively inhibited apoptosis in vivo and in vitro.
PPAR-γ is part of the receptors of the PPAR family, which is highly produced in various organs, such as the liver and adipose tissues.Research show that PPAR-γ exerts direct anti-inflammatory effects through suppression of NF-κB down-regulates its downstream pro-inflammatory factors,such as TNF-α. [39,40]It is under a protective effect on liver. [41] PPAR-γ agonists also can inhibit monocytes activation and inflammatory factors release. [42,43] Numerous studies have shown Nrf-2 and PPAR-γ can mutually activate each other, they showed synergistic protection against liver injury. [44][45][46]Our studies revealed that G4.5-COOH enhanced the expression levels of PPAR-γ thereby protecting liver function.
The PI3K/AKT/mTOR signaling pathway plays an essential role in multiple cellular processes. Latest Studies manifested that it can promote liver fibrosis by activating PI3K/AKT/mTOR signaling. [47,48] However,the effect and relationship of PPAR-γ and PI3K/AKT/mTOR signaling pathway in Cholestatic Liver disease is not certain. Through our 14 research we found cholestatic liver can significantly elevate the P-Akt P-mTOR protein expression. G4.5-COOH suppressed ANIT-induced P-Akt P-mTOR protein expression via upregulation of PPAR-γ. GW9662 specifically inhibited the expression of PPAR, which could reverse the inhibitory effect of G4.5-COOH on the expression of p-akt and p-mtor. The same results were achieved when TNF-α was used to stimulate hepatocyte in vitro.

Conclusions
G4.5-COOH PAMAM dendrimer has a certain therapeutic effect on cholestatic liver injury. It may inhibit TNF-α to damage hepatocytes through inhibition of PI3K/AKT/mTOR signaling pathway by a mechanism that might be partly dependent on up-regulated functional expression of PPAR-γ. Figure 1 The FTIR spectrum of G4.5-COOCH3 and G4.5-COOH PAMAM dendrimers.