Unraveling the hepatic stellate cells mediated mechanisms in aging's influence on liver fibrosis

Aging enhances numerous processes that compromise homeostasis and pathophysiological processes. Among these, activated HSCs play a pivotal role in advancing liver fibrosis. This research delved into how aging impacts liver fibrosis mechanisms. The study involved 32 albino rats categorized into four groups: Group I (young controls), Group II (young with liver fibrosis), Group III (old controls), and Group IV (old with liver fibrosis). Various parameters including serum ALT, adiponectin, leptin, and cholesterol levels were evaluated. Histopathological analysis was performed, alongside assessments of TGF-β, FOXP3, and CD133 gene expressions. Markers of fibrosis and apoptosis were the highest in group IV. Adiponectin levels significantly decreased in Group IV compared to all other groups except Group II, while cholesterol levels were significantly higher in liver fibrosis groups than their respective control groups. Group III displayed high hepatic expression of desmin, α-SMA, GFAP and TGF- β and in contrast to Group I. Increased TGF-β and FOXP3 gene expressions were observed in Group IV relative to Group II, while CD133 gene expression decreased in Group IV compared to Group II. In conclusion, aging modulates immune responses, impairs regenerative capacities via HSC activation, and influences adipokine and cholesterol levels, elevating the susceptibility to liver fibrosis.


Hematoxylin and eosin staining results
The examined sections of the control rats showed the normal structure of the liver lobes, with cords of polygonal acidophilic hepatocytes that had well-preserved vesicular nuclei.(Fig. 1a&b) shows the presence of narrow blood sinusoids with a thin wall between the hepatocyte cords and the endothelium lining.A bile duct is lined with cuboidal cells, and a portal vein with a thin wall is present in the portal region (Fig. 1c).
In contrast, hepatocytes in Group (II) sections underwent hemorrhagic necrosis and apoptosis.Thick collagen fibers with well-developed fibrosis and early cirrhosis were clearly visible (Fig. 1d, e, f).
In addition, Group (III) exhibited a slightly altered hepatic lobular architecture when compared to Group (I) in the form of less pronounced cell boundaries with less distinct contours of sinusoids (Fig. 2 a, b, c).In Group (IV), there was evident cellular damage characterized by the presence of thick collagen fibers, cirrhosis, and fibrosis with cell infiltration and marked intercellular spacing (Fig. 2d, e, f).

Masson trichrome (MT) results
In Group (I), extremely minimal basophilic collagen fibers surrounding the central and portal veins respectively (Fig. 3a, b).Conversely, Group (II) demonstrated a notable increase in basophilic collagen fibers surrounding the central and portal veins respectively compared with Group (I) (Fig. 3c, d).
In Group (III), there was a slight basophilic collagen fibers surrounding the central and portal veins respectively, as compared to Group (I) (Fig. 4a, b).In contrast, in Group (IV), there was an observed increase in basophilic collagen fibers compared with Group (III) (Fig. 4c, d).
Table 1.Serum assessment of ALT, cholesterol, adiponectin, and leptin in all studied groups.All continuous data were expressed using mean (± SD) where: P value ≤ 0.001** was considered statistically highly significant(S).a means: significant compared with group 1. b means: significant compared with group 2. c means : significant compared with group 3.

Immune-histochemistry results
• Desmin immune-histochemical staining In Group (I), HSCs had a normal distribution and were rarely observed in Desmin immune-reactive cytoplasmic reaction in the smooth muscle cells of blood vessel walls, in a small number of myofibroblasts of the portal stroma, and in specific cells found in zones 1 and 3 of the liver lobules (Fig. 5a, b).Conversely, Group (II) demonstrated a notable increase in the number of HSC immune-positive nuclei with increased parenchymal reaction compared with Group (I) (Fig. 5c, d).In Group (III), a slight alteration in the normal distribution of aging HSCs in myofibroblasts located in the interface zone between the portal space and the lobule, where both animal groups' stellate cell populations grew noticeably expressing desmin immune-reactive cytoplasmic reaction, as compared to Group (I) (Fig. 6a, b).In contrast, in Group (IV), there was an observed increase in the number of HSC immune-positive nuclei with increased parenchymal reaction compared with Group (III) (Fig. 6c, d).
• α -SMA immune-histochemical staining In Group (I), activated HSCs were rarely detectable in α-SMA immune reactivity with normal distribution in the wall of central vein and hepatic artery and portal vein branches (Fig. 7a, b).In contrast to Group (I), Group (II) demonstrated a noticeable increase in activated HSC immune-positive cells in the hepatic artery and portal vein branch walls and blood sinusoids (Fig. 7c, d).
The distribution of aged HSCs in Group (III) exhibited a slight alteration in the form of myofibroblasts that stained intensely for α-SMA could be easily identified, and they formed layers around the proliferated bile ductules when compared to Group (I), as demonstrated in (Fig. 8a, b).In contrast, Group (IV) exhibited more HSC immune-positive reactions than Group (III) in hepatic arteries and blood sinusoid walls (Fig. 8c, d).
In contrast to Group (I), Group (III) demonstrated widespread of numerous strong GFAP positive cells around portal vein, along sinusoids and in fibrotic lesions with increased number of aged HSCs with mild distribution in GFAP immune-reactive areas (Fig. 10a, b).However, Group (IV) exhibited an apparent increased number and intensity of GFAP positive cytoplasmic immune reaction when compared to Group (III) (Fig. 10c, d).

Histo-morphometric results
Group (II) demonstrated a statistically significant increase in the mean number of HSC/0.5 mm2, optical density (OD) of α-SMA, area percentage of GFAP, and TGF-β immune-positive expression compared to Group (I).This was evidenced by the results of the histo-morphometric analysis as presented in (Table .2).Furthermore, there was a statistically significant reduction observed in the four parameters when comparing Group (III) to Group (II).However, the comparison between Group (III) and Group (I) demonstrated a statistically significant increase in the four measures.In addition, a statistically significant increase was observed when comparing Group (IV) to Group (III).

Effect of induction of liver fibrosis on the expression levels of FOXP3, CD133, and TGF-β genes
The expression levels of FOXP3, CD133, and TGF beta genes in rats significantly increased after induction of liver fibrosis, as shown in (Table .3).We observed a substantial increase in the expression levels of FOXP3, CD133, and TGF-β genes in the liver fibrosis induced groups (II and IV) compared to the normal groups (I and III), respectively (P < 0.001).FOXP3 and TGF-β expression levels in Group IV reported the highest levels compared to Group II.Nevertheless, the expression level of CD133 was higher in Group II compared to other groups (I, III, and IV) (P < 0.001).In addition, there was no significant difference between Group III and Group I regarding all examined parameters (Table 4).

Effect of induction of liver fibrosis on hepatic tissue concentration of TGF-β and α-SMA
Regarding TGF-β and α-SMA concentrations in the hepatic tissue as illustrated in (Table 5), there was a significant increase in Group II in comparison to Group I.In addition, there was a non-significant increase in TGF-β concentration in Group III in comparison to Group I while there was significant increase in α-SMA concentration in Group III in comparison to group 1.Moreover, in Group IV there was a significant increase in TGF-β and α-SMA concentrations in comparison to group I, II and III.

Discussion
Liver Cirrhosis ranks as the 12th most common cause of death worldwide 27 .One of the primary histological consequences of chronic liver disease is the accumulation of collagen fibers, leading to progressive liver fibrosis and ultimately cirrhosis.The prevalence of liver fibrosis significantly rises with age, with men and women experiencing increased rates from their 40s and 50s, respectively.Among individuals aged 70 or older, the prevalence of significant liver fibrosis reaches 15.3% 28,29 .The process of fibrogenesis resulting from liver injury involves intricate interactions among various cell types responding to chronic inflammation, fibrogenesis, and tissue regeneration 25 .Our study, to the best of our knowledge, is the first to elucidate the complex interactome of aging in liver fibrogenesis and regeneration.While the primary function of activated HSC is the synthesis of extracellular matrix proteins, many aspects of this cell type remain unknown 30 .
In our study, we validated liver fibrosis through elevated liver enzymes and the high expression of desmin, α-SMA, GFAP and TGF-β.These findings are consistent with Jeng et al., who reported a positive correlation between α-SMA expression and the degree of fibrosis 31 , while GFAP serves as an early marker of liver fibrosis 32 , and desmin significantly increases in advanced fibrosis 33 .We observed significantly higher expression of both HSC-activation markers, GFAP and desmin, in the fibrosed groups, with group IV exhibiting greater expression compared to group II.On the same side, we made the unexpected observation of elevated expression of fibrosis markers in group III when compared to the control group I.This finding underscores the significant role that aging plays in both the onset and progression of liver fibrosis.
Recent studies have highlighted that stellate cell activation can be beneficial, as it alters gene expression and contributes to liver regeneration through the release of trophic factors 34 .Activated stellate cells are known to promote liver regeneration by releasing retinoids, which support hepatocyte survival and stimulate the release of growth factors, such as Hepatocyte Growth Factor (HGF) [35][36][37] .HSCs possess stem cell characteristics, including resistance to apoptosis, nestin expression, and CD133 expression, a marker of stem/progenitor cells 30,38 .While CD133 has been studied as a cancer stem cell marker, its role in stellate cells remains less understood 34 .In our study, we observed a significant increase in CD133 levels, as well as TGF-β, in the fibrosis groups, indicating their potential involvement in liver fibrosis 39 .
Our findings align with Rozeik et al., as we also observed a significant increase in CD133 expression with the inflammatory activity scores and stages of liver fibrosis 40 .However, group II exhibited higher CD133 expression compared to group IV, suggesting a greater regenerative capacity in the former 41 ..It was discovered that retinoic acid (RA) controlled the progression of the cell cycle and sped up liver regeneration.This was linked to an early activation of (RA) signaling, as well as elevated expression of receptor binding proteins and retinoid processing enzymes 42 .
The aging process affects the stellate cell niche, potentially explaining its impact on liver fibrosis and regeneration 37 .Aged stellate cells exhibit a senescence-associated secretory phenotype and reduced expression of GFAP.These aging-related changes result in the release of HSCs from their niche and impair the functions of stellate cells by reducing the expression of ECM-associated genes and growth factors like HGF.These factors collectively contribute to the diminished regenerative capacity of aged livers 34 .
The interaction between the immune system and stellate cells is complex, with HSCs displaying immunomodulatory properties similar to mesenchymal stem cells from the bone marrow.In early-stage fibrosis, immune responses contribute to terminating HSC activation, but the interaction of immune mediators in chronic liver disease frequently encourages liver fibrosis 43 .
The activation of Tregs cells during aging is a notable alteration in the adaptive immune response.Tregs has an immunosuppressive function, promoting immune self-tolerance and homeostasis by secreting interleukin-10 (IL-10) and TGF-β.The transcription factor FoxP3 is a distinctive marker of Treg cells and is essential for both the development of Treg and immune control 44 .In our study, we found a selective increase in Treg cells expressing Foxp3 + in the aged fibrotic group compared to the young fibrotic one, and both fibrotic groups are higher than other control groups.This observation is consistent with several previous research [45][46][47] .
In the present study, we also observed significantly increased levels of Foxp3 and TGFβ in the old fibrotic groups.These findings are in line with the study by Lee et al., which demonstrated that cytokines like IL-10 and TGF-β accumulate with age and induce the transformation of CD4 + CD25-FOXP3-T cells into CD4 + CD25 + FOXP3 + Tregs, which suppress the activation and proliferation of CD4 + and CD8 + T cells 48 .
Aging is associated with changes in the proliferative and functional abilities of the immune system, leading to immunosenescence and inflammation.Th17 cells have been implicated in autoimmune and chronic inflammatory diseases, with a reciprocal relationship between Th17 cells and Tregs 49 .The Treg/Th17 ratio plays a critical role in intrahepatic immune regulation, and its dysregulation is characteristic of liver fibrosis progression.Schmitt et al. found that the Treg/Th17 ratio decreases with age 49 .
On the other hand, some studies have reported no significant change in Treg cell frequencies during aging, but the capacity of CD4 + FOXP3 + T cells to regulate IL-10 production decreases 50 .There is potential for establishing We observed in our study that elevated cholesterol levels were accompanied by increased liver enzyme (ALT) levels, particularly in the older fibrosed groups compared to their controls.Aligning with this finding, it has been reported that age-related glucose intolerance in humans often coincides with the development of insulin resistance 51 which serves as one of the underlying mechanisms contributing to elevated levels of very low density lipoprotein (VLDL) cholesterol and low density lipoprotein (LDL) cholesterol.The excessive production of VLDL and triglycerides within the liver has been postulated to stem from elevated serum levels of free fatty acids 52 .
Teratani et al. have previously documented that the accumulation of free cholesterol in HSCs can lead to heightened Toll-like receptors 4 (TLR4) signaling, rendering HSCs more sensitive to TGF-β 53 .Furthermore, cholesterol can activate HSCs through various mechanisms, such as the activation of cholesterol crystal-activated Kupffer cells and oxidized LDL-loaded foam cells, which can induce HSC activation through the release of inflammatory cytokines 54 .Recent research has also demonstrated that oxidized phospholipids can directly trigger the expression of fibrogenic genes in HSCs 55 .Conversely, the study conducted by Kaminsky-Kolesnikov suggests that cholesterol may promote hepatocyte proliferation to mitigate liver scarring by inducing apoptosis in activated HSCs 56 .
Adiponectin, one of the most abundant adipokines, exhibits hepatoprotective and antifibrogenic effects during liver injury.These effects include inhibiting the production of IL-6 and Tumor necrosis factor-α (TNF-α) and HSC proliferation and migration 57 .Our study revealed a significant reduction in adiponectin levels in group IV, characterized by high cholesterol levels and advanced fibrosis, compared to group II.Mohamed et al. and Nazal et al. similarly found a highly significant negative correlation between adiponectin levels and liver fibrosis, with significantly lower adiponectin levels in individuals with high-grade fibrosis 57,58 .
Our study further demonstrated a decrease in adiponectin levels in the older group IV compared to the younger group II, a phenomenon that may be attributed to the presence of insulin resistance, as suggested by Yamauchi et al. 59 .
Several recent reports have highlighted the role of leptin as a critical hormone in the development of liver fibrogenesis, primarily by enhancing the activity of TGF-β1 60 .
The study by Saxena et al. in 2002, investigating the role of leptin in hepatic fibrosis, found that the absence of leptin led to the absence of α-SMA positive cells, underscoring the importance of leptin as a profibrogenic factor in liver fibrosis 61 .Our study also indicated a decreased level of leptin in group IV compared to group II.This decrease may be attributed to aging-related factors, such as the down-regulation of megalin expression, www.nature.com/scientificreports/reduced leptin uptake in the hypothalamus due to decreased expression of leptin receptor mRNA, and decreased levels of leptin receptor protein in the hypothalamus, as outlined by Sasaki in 2015 62 .

Conclusion
Aging had a critical role in the enhancement of liver fibrosis, and there are many underlying mechanisms that may promote this role including and not limited to hormonal changes like adipokines, decreased regenerative capacities and immunity modulation.Hepatic stellate cells were engaged effectively in most of these mechanisms, so we recommend further studies to investigate the crucial intermediate role of hepatic stellate cells as a potential therapeutic target.

Experimental animals
The animals utilized were female Albino rats, weighing 200-230 g each.Standard conditions were prepared for rats to keep them in a temperature (25 ˚C) with 12 h of light and 12 h of darkness and unrestricted access to standard laboratory food and water.This study has been approved by the ethics committee of The Institutional Animal Care and Use Committee, Zagazig University (ZU-IACUC), Egypt (ZU-IACUC /2/F/64/2022).All the procedures and experiments were carried out in accordance with the protocol approved by them, and the animals were handled according to the national and international ethical standards.To keep the experimental animals from experiencing pain or discomfort, this study utilized the earliest endpoint that was scientifically justified.All experiments were performed in accordance with ARRIVE guidelines and regulations.

Experimental design
Four groups of total 32 albino rats were randomly assigned (8 animals per group): Group I (young "3 months old" with normal liver, n = 8), Group II (young "3 months old" with liver fibrosis, n = 8), Group III (old "18 months old" with normal liver, n = 8) and Group IV (old "18 months old" with liver fibrosis, n = 8).In group II and group IV, induction of fibrosis was done by treating rats two times a week for 8 weeks with thioacetamide (200 mg/kg, ip) (Sigma, St. Louis, MO) 63 .Blood samples were taken from all groups twenty-four hours after the last dose of medication, and serum was isolated and kept at-20 ˚C for further investigations.Then, rats were anesthetized by intraperitoneal injection of Pentobarbital sodium 50mg/kg and sacrificed for collection of liver samples.

Serum analysis for detection of cholesterol, adiponectin, leptin and ALT
• Measurement of serum cholesterol level The serum cholesterol levels were detected by using the commercial kit (SPINREACT, Spain) based on the manufacturer's instructions.The intensity of the color formed was measured, which is proportional to the cholesterol concentration in the sample.

• Measurement of serum adiponectin and leptin levels
The quantitative determination of rat adiponectin (ADP) and leptin (LEP) concentrations in serum was performed using sandwich ELISA technique as directed by the instructions (CUSABIO rat adiponectin (ADP) ELISA Kit, USA; Catalog No. CSB E07271r) and (CUSABIO rat leptin (LEP) ELISA Kit, USA; Catalog No. CSBE07433r).The assay ranges were 0.156 ng/ml-10ng/ml and 0.06 ng/ml-50 ng/ml, respectively.
• Measurement of serum ALT Levels of serum ALT were determined using the commercially available kit (SPINREACT, Spain) in accordance with the manufacturer's guidelines.Using photometric measurement, the rate of decline in NADH concentration was determined.It is correlated with the amount of ALT present in the sample that is catalytically concentrated.

Tissue sampling
The rats underwent an overnight fasting by the end of the experiment.The livers were properly dissected, rapidly taken from each group, maintained on ice-cold plates for thirty minutes, and dried after they had been anaesthetized with intraperitoneal injection of Pentobarbital sodium 50mg/kg 64 .All samples from each group were preserved in Bouin's solution and prepared as paraffin blocks for inspection under a light microscope.The Pathology Department, Faculty of Medicine, Zagazig University, was the site of all procedures.As per routine practices 65 , sections were cut on 5µm thickness, they were dewaxed in xylene, rehydrated in descending grades of alcohol then were stained by H&E.
b Fibrosis Assessment using Masson Trichrome (MT) stain The Pathology Department, Faculty of Medicine, Zagazig University, was the site of all procedures.As per routine practices 65 , Masson trichrome (MT) stain was used to stain sections that were 5 µm thick that had been placed on glass slides and deparaffinized in xylene.
c Immunohistochemistry (IHC) for Desmin, α-SMA, GFAP & Transforming Growth Factor -β (TGF-β): Desmin, a HSC marker, as well as α-SMA, a marker for activated HSCs, were stained.The liver samples utilized were set in paraffin blocks.Desmin and α-SMA were stained in sections from each block using the Novocastra Novocastra (NCL-DER11 and Novocastra (NCL-SMA) dilutions of 1:200 each).On a Leica Bond Polymer, Refine Detection (DS9800) with Bond DAB enhancer 30 mL (AR9432), rat liver sections were treated according to an automatic IHC Leica staining protocol.For the antigen retrieval stage, which lasted 30 min, EDTA buffer pH 9 was employed to detect desmin.Sections were pretreated with 0.3% hydrogen peroxide for 20 min in PBS and avidin-biotin blocking solutions then overnight primary antibody incubation at 4°C was done.The antimouse biotinylated secondary antibody (1: 150 Sigma, St. Louis, MO) had been applied for 40 min at room temperature then the peroxidase-conjugated streptavidin (1: 50 Sigma, St. Louis, MO) was administered.3,3-Diaminibenzidine was used to demonstrate peroxidase activity 66 .For the biotinylated monoclonal mouse antibody for GFAP and the polyclonal anti-mouse antibody for TGF-β, the primary antibody was diluted using PBS at dilutions of 1/500 and 1/100, respectively.The slides were then rinsed with PBS for 5 min before drops of streptavidin peroxidase were applied for 20 min.The slides were chromogenated with diaminobenzidine then treated with distilled water.The slides were then dried, stained with Harris hematoxylin, and cleaned in xylene.Brown color indicated a favorable response.PBS was used as a negative control in place of the main antibody.Universal kits 67 produced by Biogen Inc. (Cambridge, Massachusetts, USA) for both GFAP and TGF-β were obtained from Dako Company (Egypt).

Histo-morphometric analysis
A morphometric analysis was performed on each group's rats.At a magnification of 400, 10 non-overlapping fields were captured and examined.Manual counting was done on desmin-positive nucleated cells for each image/0.5mm2.After immunostaining, the density of α-SMA immune-reactive and parenchymal area was determined (large vessels were eliminated) were assessed using optical density (OD), which was acquired by transforming the mean gray level using the following formula: OD = log (256/mean gray level).Following the subtraction of the background, a ratio of the OD of image file was calibrated as % (relative optical density, ROD) using Image J analysis software (Fiji image j; 1.51 n, NIH, USA) at human anatomy and embryology department, Zagazig University.On the other hand, in GFAP and TGF-β immunohistochemical stained sections, to quantify the percentage area of positive response from 8 rats/group, perceptual fields across the pictures captured by the light microscope at 400 × amplification were selected.To perform a quantitative analysis of the immunoreactivities, stained sections were examined and analyzed by light microscopy (LEICA ICC50 W) in Image Analysis Unit of Human Anatomy and Embryology Department, Faculty of Medicine, Zagazig University.

Gene expression assessment for FOXP3, TGF-ß and CD133 using real time PCR Total RNA extraction from tissue
Total RNA was extracted from homogenized liver tissue using Trizol (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with manufacturing data.To evaluate the RNA quality, the A260/A280 ratio was analyzed using the NanoDrop® ND-1000 Spectrophotometer (NanoDrop Technologies; Wilmington, Delaware, United States) for 1.5l of the RNA.The estimated purity used for any given RNA was between 1.8 and 2.0.High-Capacity cDNA Reverse Transcription Kit cDNA Kit; (Applied Biosystems™, USA) was used for cDNA synthesis from mRNA extracted in total volume 20 μl as a total of 9 μl containing (1 μg) of RNA sample was added to 11 μL of 2X reverse transcriptase (RT) master mix.then incubated for 60 min at 45 °C, followed by incubation for 10 min at 85 °C Table 3. Expression levels of FOXP3, CD133, and TGF-β genes in all studied groups.All continuous data were expressed using mean (± SD) where: P value ≤ 0.001** was considered statistically highly significant(S).a means : significant compared with group 1. b means: significant compared with group 2. c means : significant compared with group 3.

Table 4 .
Primers used in qRT-PCR.Table.5.Hepatic tissue concentration of TGF-β and α-SMA in all studied groups.All continuous data were expressed using mean (± SD) where: P value ≤ 0.001** was considered statistically highly significant (S). a means: significant compared with group I. b means significant compared with group II.c means: significant compared with group III.