Genistein was obtained from Dalian Meilun Biology Technology Co., Ltd. (Dalian, China). Dimethylnitrosamine was obtained from TCI (Shanghai) Chemical Industry Development Co., Ltd. Dulbecco’s modified Eagle’s medium (DMEM, high glucose) and fetal bovine serum (FBS) were obtained from Gibco Life Technology (Gibco Invitrogen Corporation, Barcelona, Spain). Aspertate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and total bile acid (TBA) test kits, hematoxylin and Eosin (H&E) staining kit, and hydroxyproline (Hyp) assay kit were obtained from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Reverse transcriptase assay kit was purchased from Applied Biological Materials Inc. (abm, Vancouver, Canada). TRIzol reagent and all primer were obtained from Shanghai Sangon Biological Engineering Co. Ltd. (Shanghai, China). SYBR Green Realtime PCR master mix was obtained from Toyobo Co., Ltd. (Osaka, Japan) Cell counting kit-8 (CCK-8) was obtained from Dojindo Laboratories Co., Ltd. (Kumamoto, Japan). BeyoClick™ EdU cell proliferation kit with Alexa Fluor 594 and cell cycle and apoptosis analysis kit were obtained from Beyotimebiology Co., Ltd. (Shanghai, China). RIPA lysis buffer, proteinase and phosphatase inhibitor, BCA protein concentration assay kit and QuickBlock™ blocking buffer for western blot were obtained from Beyotimebiology Co., Ltd. (Shanghai, China). Antibodies JAK2 (#3230), p-JAK2 (#3771), STAT3 (#9139) and p-STAT3 (#9145) were obtained from Cell Signaling Technology (Danvers, MA, United States); $\alpha$-smooth muscle actin ($\alpha$-SMA, #ab7817, for Western blot; #ab124964, for immunofluorescence), collagen type I alpha 1 (Col1A1, #ab34710), SOCS3, and CD68 (#ab125212), as well as Goat Anti-Rabbit IgG H&L (Cy3 ®) preadsorbed (#ab6939) were obtained from Abcam, Inc. (Cambridge, UK). CD163 (#16646-1-AP), CD206 (#60143-1-Ig) and GAPDH (#60004-1-Ig) were purchased from Proteintech (Wuhan, China). HSC line LX2 cells was provided by Professor Lieming Xu.

Forty male Wistar rats (SPF grade, weight of 140 $\pm$ 20 g) were provided by Shanghai Xipuer-Bikai Experimental Animal Co., Ltd. Animal experiment was carried out in Experimental Animal Center of Shanghai University of Traditional Chinese Medicine. The animal study was reviewed and approved by the Animal Ethics Committee of Shanghai University of Traditional Chinese Medicine (PZSHUTCM190322007; Approval date: July 27, 2018). All rats (weighing 160–180 g) were randomly divided into the normal control (n = 8) and liver fibrosis model group (n = 32). An experimental model of liver fibrosis was established by intraperitoneal injection of 0.5% DMN saline (2 mL/kg body weight, 3 consecutive days per week) for 4 weeks according to the previous study [23]. After the second week, 32 rats with liver fibrosis were further divided into four groups with eight rats in each group as follows: DMN model, genistein-5 mg/kg, genistein-20 mg/kg, and sorafenib (5 mg/kg, as a positive control drug) group. From weeks 3 to 4, rats received treatment with 0.4% CMC-Na or correspondence drugs by gavage per day. After the fourth week, all rats were anesthetized by 3% pentobarbital sodium through intraperitoneal injection, and the samples were collected for further studies.

LX2 cells were seeded in 12-well plates with 5 $\times$ 10${}^5$ cells and 30 mm petri dishes with 2 $\times$ 10${}^6$ cells and were maintained in 10% FBS-containing DMEM medium. The cells were incubated with genistein at concentrations of 20, 40, and 80 $\mu$M. The divided experimental groups were as follows: DMSO control group, TGF-$\beta$1 (5 ng/mL) group, TGF-$\beta$1 + SB431542 (10 $\mu$M, an TGF-$\beta$1 inhibitor) group, TGF-$\beta$1 + 20 $\mu$M genistein group, TGF-$\beta$1 + 40 $\mu$M genistein group, TGF-$\beta$1 + 80 $\mu$M genistein group. The mRNA and protein expressions were evaluated by qRT-PCR, Western blot and immunofluorescent assay.

LX2 cells were cultured at 5 $\times$ 10${}^3$ cells/well into 96-well plate and incubated in 10%-containing DMEM medium at appropriate times. Subsequently, cells were incubated with genistein at concentration of 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 $\mu$M. And then, 10 $\mu$L CCK-8 solution was quickly added to each well at two hours before the scheduled times (24 h). Ultimately, the absorbance of 96-well plates was detected at 450 nm by microplate reader (BioTek, Hercules, CA, USA) at the scheduled times.

LX2 cells were grown in 12-well plates and were treated with genistein (20, 40 and 80 $\mu$M) for 24 h. Then, the cells were incubated with EdU labeling medium with a final concentration of 10 $\mu$M for additional 2 h at 37 ${}^{\circ }$C. Two hours later, the cells were fixed with 4% formaldehyde for 10 min, were permeabilized with 0.3% Triton X-100 for 15 min, and were stained with click reaction solution for 30 min, respectively. The cell nuclei were stained using hoechst for 10 min. Finally, the cells were observed using an inverted fluorescence microscope DP80 (Olympus, Tokyo, Japan).

Briefly, LX2 cells, seeded into the 6-well plate at 1 $\times$ 10${}^5$ cells per well, were cultured with genistein (20, 40 and 80 $\mu$M) for 24 h. The cells were then washed using ice-cold PBS, harvested and fixed in EP tube with cold 70% ethanol for 24 h, and followed by staining with PI for 30 min 37 ${}^{\circ }$C, respectively. Added 500 $\mu$L propidium iodide staining solution and incubated for 30 min at 37 ${}^{\circ }$C in the dark. Finally, the samples were analyzed by flow cytometry (FACS AriaIII, BD, USA). The percentage of cells in different phases of the cell cycle was analyzed by ModFit LT 5.0 software (Verity Software House, Inc., Topsham, ME, USA).

Serum AST, ALT, ALP and TBA levels, and the content of hepatic Hyp were measured using a matched test kit. In short, the serum samples were thawed at room temperature, and then placed in an automatic analyzer according to the labeling sequence. Subsequently, the corresponding detection reagents were added into the analyzer detection reagent bottle to detect serum AST, ALT, ALP, and TBA levels. Furthermore, hepatic Hyp content was assessed by alkaline hydrolysis method. Approximately 50 mg of liver tissues were weighed and hydrolyzed, and pH was adjusted to 6.0–6.8. The detection steps were performed according to the manufacturer’s instructions.

Formalin-fixed paraffin-embedded tissue samples were cut into 4-$\mu$m sections, and then adhered to silanized slides. The samples were then stained by H&E and Sirius red staining to evaluate histopathological features and fibrosis stage according to manufacturer’s instructions. Sirius red-positive area was further calculated by Leica LAS Image Analysis, percentage of positive staining area = positive staining area/total area $\times$ 100%.

The tissue sections were routinely de-waxed and dehydrated, and underwent antigen retrieval with mixing 4% sodium citrate buffer. Endogenous peroxidase activity and non-specific antigens were blocked with methanol containing 3% H${}_2$O${}_2$ for 15 min and PBS containing 10% goat serum for 30 min, respectively. Further, the sections were incubated with anti-$\alpha$-SMA (1:1000), anti-Col1A1 (1:500), anti-CD68 (1:1000), anti-CD163 (1:1000), and anti-CD206 (1:1000) at 4 ${}^{\circ }$C overnight. PBS was used instead of the primary antibody as the negative control. The following day the sections were incubated using secondary antibody labeled with HRP. Finally, the sections were stained by 3, 3-diaminobenzidine (DAB) and harris hematoxylin.

An 8 $\mu$m-thick frozen sections of liver tissues were fixed with cold acetone for 10 min. The cells were fixed with 4% formaldehyde for 15 min and were permeabilized with 0.3% Triton X-100 for 10 min. Then, fixed tissue sections and cells were blocked with 10% goat serum for 30 min, followed by incubation with anti-$\alpha$-SMA (1:1000) and anti-Col1A1 (1:500) overnight at 4 ${}^{\circ }$C, respectively. Meanwhile, PBS was used instead of the primary antibody as the negative control. Subsequently, the specimens were stained using fluorescent secondary antibodies with goat anti-rabbit IgG-Cy3 (1:3000) and the nuclei were counterstained with DAPI (1:1000). Finally, these specimens were scanned under Olympus Fluoview 500 confocal microscope (Malvern, USA), and representative images were displayed.

The proteins from the liver tissues and LX2 cells were extracted using RIPA lysis buffer. Simply, LX2 cells and liver tissues were lysed with RIPA lysis buffer containing phosphatase and protease inhibitor, and lysed tissues and cells were centrifuged at 12,000 rpm for 20 min. The protein concentration was measured using the BCA protein assay kit. And then, the samples, SDS-PAGE protein loading buffer (5$\times$) and water were mixed together to prepare loading protein samples. Subsequently, 30 $\mu$g proteins were loaded on 8% or 12% SDS-PAGE (electrophoresis conditions: 80 V) and transferred onto 0.45 $\mu$$M$ polyvinylidene fluoride (PVDF) membranes (transfer conditions: 2–2.5 h, 100 V). The membranes were blocked with quick sealing fluid for 30 min, and then incubated with anti-$\alpha$-SMA (1:1000), anti-JAK2 (1:500), anti-p-JAK2 (1:500), anti-STAT3 (1:500), anti-p-STAT3 (1:500), anti-SOCS3 (1:500), and anti-GAPDH (1:5000) at 4 ${}^{\circ }$C overnight and subsequently incubated with fluorescence-labeled secondary antibody (1:5000). The protein bands were scanned by the Odyssey infrared scanner (LI-COR Biosciences, Lincoln, NE, USA).

Total RNA from rat hepatic tissues and LX2 cells was extracted using TRIzol reagent. Briefly, liver tissues and LX2 cells were lysed using TRIzol reagents. Afterward, chloroform, isopropanol, 75% ethanol, and anhydrous ethanol were added to the solutions of homogenized tissues and lysed cells, and then centrifuged at 12,000 rpm 4 ${}^{\circ }$C and finally dissolved with DEPC water. cDNA was synthesized from extracted total RNA using the reverse transcriptase PCR kit. qRT-PCR was carried out using SYBR Green PCR master mix. The relative mRNA expression levels of liver tissues and LX2 cells were measured using the ABI ViiA7 sequence detector (Applied Biosystems, USA). The PCR cycling program was 95 ${}^{\circ }$C for 60 s, 40 cycles of 95 ${}^{\circ }$C for 15 s, and 60 ${}^{\circ }$C for 60 s. Gene expression was normalized to that of the housekeeping gene GAPDH. The relative expression of target genes was calculated using 2${}^{-\mathrm{\Delta }\mathrm{\Delta }Ct}$ method. The primer sequences for qRT-PCR are listed in Table 1.

Data are expressed as mean $\pm$ standard deviation (SD). Multiple groups mean with normal distributions and equality of variance were compared by one-way ANOVA with post hoc LSD for multiple pair-wise comparisons. Non-normal distributions and unequal variance were analyzed using Kruskal-Wallis test. p 0.05 was considered statistically significant.

Genistein was used in HSC line LX2 cells to evaluate the effect of genistein on cell growth by CCK8 assay, EdU staining, cell cycle analysis. First, the cell viability was evaluated by CCK8 assay, compared with the control group, cell viability was not significantly inhibited within 150 $\mu$$M$, but was obviously inhibited by genistein treatment with 200 $\mu$$M$ (p 0.05, Fig. 2A). Further, the anti-proliferative effect of genistein was investigated in LX2 cells by EdU staining, the results showed that the proliferation of LX2 cells was significantly inhibited at 40 and 80 $\mu$$M$ of genistein (p 0.05 or p 0.001, Fig. 2B). The cell cycle distribution was evaluated using flow cytometry. In comparison with the control group, the G0/G1 phase population increased, and the S phase population decreased in LX2 cells after treatment with 20, 40, and 80 $\mu$$M$ genistein (p 0.05 or p 0.01 or p 0.001), suggesting that genistein induced cell cycle arrest at G0/G1 phase (Fig. 2C).

Fig. 2.

The effects of genistein on cell viability, proliferating and cell cycle. (A) CCK8 test was used to evaluate the effect of different concentrations of genistein on cell viability at 24 h in LX2 cells was detected by. (B) EdU assay was performed to detected the effect of genistein (20, 40 and 80 $\mu$$M$) on the proliferation at 24 h in LX2 cells, and the number of positive cells in EdU assay, Hoechst was counterstained the nuclei, representative confocal microscopy images are shown, scale bar 20 $\mu$m. (C) Flow cytometry was used to evaluate the effect of genistein (20, 40 and 80 $\mu$$M$) on the cell cycle at 24 h in LX2 cells. ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001, ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01,${}^{\mathrm{\#}}$p 0.05 vs control group (0.1% DMSO).

HSC activation is a key event in the occurrence and development of liver fibrosis. Thus, its activation is usually evaluated in the study of fibrosis. In this work, the expressions of $\alpha$-SMA and Col1A1, which are typical markers of HSC activation, were induced by TGF-$\beta$1 in LX2 cells to investigate the anti-fibrosis effects of genistein in vitro. First, the expressions of $\alpha$-SMA and Col1A1 mRNA were assessed by qRT-PCR analysis. As expected, the elevated mRNA expression levels of $\alpha$-SMA and Col1A1 in LX2 cells induced by TGF-$\beta$1 were decreased after treatment with 20, 40, and 80 $\mu$M of genistein (p 0.05 or p 0.01 or p 0.001; Fig. 3A). The protein expression of $\alpha$-SMA was also evaluated by Western blot assay and immunofluorescent staining. Western blot assay result showed that the expression of $\alpha$-SMA protein was significantly suppressed in genistein treatment group (p 0.05 or p 0.001; Fig. 3B). Consistent results of $\alpha$-SMA protein expression were achieved by immunofluorescence staining (Fig. 3C).

Fig. 3.

The effects of genistein on the expressions of $\alpha$-SMA and Col1A1 in LX2 cells. (A) qRT-PCR was used to determine the effects of genistein on the expression levels of $\alpha$-SMA and Col1A1 mRNA in LX2 cells, gene expression was normalized to GAPDH mRNA. (B) Western blot was used to assess the effect of genistein on the expression level of $\alpha$-SMA protein, $\alpha$-SMA protein expression was normalized against GAPDH level. (C) Immunofluorescence staining of $\alpha$-SMA was assessed by immunofluorescence assays, DAPI was counterstained the nuclei, representative confocal microscopy images are shown, scale bar 60 $\mu$m. ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs control group (0.1% DMSO);${}^{***}$p 0.001, ${}^{**}$p 0.01, ${}^{*}$p 0.05 vs TGF-$\beta$1 group. SB43152 is an inhibitor of TGF-$\beta$1.

Liver pathological histology was determined in rats by H&E staining. As shown in Fig. 4A, the structure of hepatic lobular was severely collapsed and formed more complete pseudo-lobules, an abundance of inflammatory cells infiltrated in the portal tract area, the central vein area, and the hepatic sinusoid in DMN rats compared with the control rats. However, compared with the DMN rats, these changes of liver histopathology in genistein-treated rats were significantly improved. Serum biochemistry indices: AST, ALT, ALP, and TBA levels, were also detected. As shown in Fig. 4C, the serum levels of AST, ALT, ALP, and TBA were increased significantly by administration with DMN (p 0.001) and were evidently reduced by genistein treatment (p 0.05 or p 0.01). However, there was no significant difference in sorafenib group, compared with the DMN group.

Fig. 4.

The effects of genistein on the liver injury and the collagen deposition in the DMN-induced rats. (A) Representative images of H&E staining (scale bar 100 $\mu$m). (B) Representative histological images stained with sirius red staining (scale bar 200 $\mu$m). (C) Serum levels of ALT, AST, ALP and TBA were measured in rats. (D) Sirius red-positive area (%) was assessed by quantitative imaging of sirius red staining. (E) Hyp content in liver was determined. ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001 vs control group. ${}^{***}$p 0.001, ${}^{**}$p 0.01,${}^{*}$p 0.05 vs DMN group. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bile acids (TBA), hydroxyproline (Hyp).

In addition, the deposition of collagen fibers was evaluated by sirius red staining. Compared with the control rats, several collagen fibers formed fibrous septum, destroyed the structure of hepatic lobule, and formed pseudo lobule in the liver with hepatic fibrosis (Fig. 4B). After genistein treatment, several changes were improved. For instance, reduced collagen depositions and fibrous septa were observed in these rats compared with the DMN-induced rats. Further, sirius red-positive area was calculated to evaluate indirectly hepatic collagen content. The result indicated that sirius red-positive area was obviously decreased after genistein treatment, compared with the DMN model group (p 0.05 or p 0.01; Fig. 4D). The liver tissue Hyp content was further measured to estimate directly the collagen deposition in the liver. The results were consistent with sirius red-positive area (p 0.01 or p 0.001; Fig. 4E).

Consistent with the in vitro experiments, the expressions of $\alpha$-SMA and Col1A1 were also evaluated in vivo. As shown in Fig. 5A–C, at the protein levels, the expressions of $\alpha$-SMA and Col1A1 were evaluated by immunohistochemistry, immunofluorescent, and Western blot assay. As expected, immunohistochemistry and immunofluorescence results indicated that the expressions of $\alpha$-SMA and Col1A1 were increased significantly in the liver with DMN-induced rats and decreased significantly in the genistein group (p 0.05 or p 0.01 or p 0.001). Similar results were obtained by Western blot assay, in which genistein could clearly reduce the expression of $\alpha$-SMA (p 0.05; Fig. 5D–F). The mRNA expressions of $\alpha$-SMA and Col1A1 at transcription levels were further confirmed by qRT-PCR, showing a significant increase in the DMN model group (p 0.001) but a remarkable reduce in the genistein group (p 0.05 or p 0.01 or p 0.001; Fig. 5G).

Fig. 5.

The effects of genistein on the expressions of HSC, MMPs and TIMPs in the DMN-induced rats. (A,B) Immunohistochemistry assay was used to determine the effects of genistein on the protein expression levels of $\alpha$-SMA and Col1A1 in the liver (scale bar 100 $\mu$m). (C) Immunohistochemistry quantification of $\alpha$-SMA and Col1A1 protein with positive area. (D,E) Immunofluorescence assay was performed to determine the effects of genistein on the protein expression levels of $\alpha$-SMA and Col1A1 in the liver (scale bar = 100 $\mu$m), the nuclei were counterstained with DAPI. (F) Western blot assay was applied to assess the protein expression level of $\alpha$-SMA in the liver, protein expression was normalized against GAPDH level. (G) qRT-PCR was performed to evaluate the effects of genistein on the expression levels of Col1A1 and $\alpha$-SMA mRNA in the liver, the expression levels of genes were normalized by GAPDH mRNA. (H) qRT-PCR was performed to assess the effects of genistein on the expression levels of MMP9, MMP2 and TIMP1 in liver, the expressions of genes were normalized by GAPDH mRNA. ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001,${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs control group.${}^{***}$p 0.001, ${}^{**}$p 0.01, ${}^{*}$p 0.05 vs DMN group.

MMPs and TIMPs are directly involved in the balance between synthesis and degradation of ECM in the liver [24]. Thus, in the present study, MMP2, MMP9, and TIMP1 at the levels of transcription were detected. The results indicated that the elevated mRNA expressions of MMP2 and MMP9 in DMN-induced rats were reversed after treatment by genistein (p 0.05; Fig. 5H). The expression of TIMP1 mRNA was also enhanced with DMN administration (p 0.001) but decreased in the genistein treatment group (p 0.05; Fig. 5H).

Liver fibrosis is an inflammatory response. Thus, inflammation related-factors were evaluated at the transcriptional levels in this work. qRT-PCR results indicated that the increased mRNA expressions of IL-1$\beta$, IL-6, TNF-$\alpha$, and MCP-1 in DMN-induced rats were decreased significantly by genistein treatment (p 0.05 or p 0.01; Fig. 6A). We investigated whether genistein could affect the distribution of both subtypes of macrophage in the liver. The expression levels of CD68 (M1 macrophage marker), CD163 and CD206 (M2 macrophage marker) in the liver were assessed by immunofluorescence staining. As shown in Fig. 6B–E, the expression of CD68 was evidently increased, but those of CD163 and CD206 were clearly decreased in the DMN-treated rats (p 0.001). After genistein treatment, the expression of CD68 was markedly decreased, and those of CD163 and CD206 increased (p 0.05 or p 0.01 or p 0.001).

Fig. 6.

The effects of genistein on inflammatory infiltration and macrophage functional properties in rats induced by DMN. (A) qRT-PCR was performed to evaluate the effects of genistein on the mRNA expression levels of IL-1$\beta$, TNF-$\alpha$, IL-6 and MCP-1 in the liver, the expression of genes were normalized by GAPDH mRNA. The expression levels of CD68 (B), CD163 (C) and CD206 (D) protein were determined by immunohistochemistry (scale bar 100 $\mu$m). (E) Immunohistochemistry quantification of CD68, CD163 and CD206 protein with positive area. ${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001, ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01, ${}^{\mathrm{\#}}$p 0.05 vs control group; ${}^{***}$p 0.001, ${}^{**}$p 0.01,${}^{*}$p 0.05 vs DMN group.

JAK2/STAT3 and SOCS3 pathway plays an important role during liver fibrosis. In the present study, the protein expressions of JAK2, STAT3, and SOCS3 and the phosphorylation of JAK2 and STAT3 were detected. In vivo, the results indicated that the expression levels of p-JAK2/JAK2 and p-STAT3/STAT3 were clearly increased in rats with DMN-induced fibrosis (p 0.01, p 0.001), but genistein could depress the expressions of p-JAK2/JAK2 and p-STAT3/STAT3 (p 0.01, p 0.001; Fig. 7A). The expression of SOCS3 protein was also upregulated in DMN-treated rats (p 0.001). After genistein treatment, the expression of SOCS3 protein was significantly inhibited (p 0.05; Fig. 7A). Interestingly, consistent results were also obtained in TGF-$\beta$1-activated LX2 cells in vitro (Fig. 7B).

Fig. 7.

The effects of genistein on the JAK2/STAT3/SOCS3 pathway. Western blot was used to assess the protein expression levels of p-STAT3, STAT3, p-JAK2, JAK2, and SOCS3 (A) in the liver and (B) in LX2 cells were assessed by, protein expression was normalized against GAPDH level. Data are shown as mean $\pm$ SD.${}^{\mathrm{\#}\mathrm{\#}\mathrm{\#}}$p 0.001, ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 vs control group; ${}^{***}$p 0.001, ${}^{**}$p 0.01, ${}^{*}$p 0.05 vs DMN/TGF-$\beta$1 group.