Honokiol attenuates ductular reaction, regulates of bile acids metabolism, and inhibits inflammatory response in murine cholestatic liver injury model

2.1 Materials

HNK was purchased from Selleck (Shanghai, China). DDC was purchased from Sigma–Aldrich (St. Louis, USA). Control diets (grain-based rodent diet, 4.5% fat) and control diets supplemented with 0.1% DDC were both purchased from Trophic Animal Feed High-Tech Co. Ltd (Jiangsu, China). BAs, including ursodeoxycholic-2,2,4,4-d4 acid (UDCA-D4, 904171), and lithocholic 2,2,4,4-d4(LCA-D4, 589349), cholic acid (CA, C1129), α-Muricholic acid (α-MCA, 700232P), β-muricholic acid (β-MCA, SML2372), chenodeoxy cholic acid (CDCA, C9377), glycocholic acid (GCA, G2878), taurocholic acid (TCA, T4009), tauro-α-muricholic acid (T-α-MCA, 700243P), tauro-β-muricholic acid (T-β-MCA, 700244P), tauro-chenodeoxycholic acid (TCDCA, T6260), deoxycholicacid (DCA, D2510), ursodeoxycholic acid (UDCA, U5127), lithocholic acid (LCA, L6250), ω-muricholic acid (ω-MCA, 700231P), taurodeoxycholic acid (TDCA, T0875), tauroursodeoxycholic acid (TUDCA, T0266), taurolithocholic acid (TLCA, T7515), tauro-ω-muricholic acid (T-ω-MCA, 700245P) were purchased from Sigma–Aldrich (St. Louis, USA). All other chemicals were obtained from commercial sources.

2.2 Chronic cholestasis liver injury model

The male mice were purchased from the Shanghai Sipple Bikai Laboratory Animal Co., Ltd (Shanghai, China). All mice were kept under specific pathogen free conditions in Shanghai University of Traditional Chinese Medicine (Shanghai, China). After all mice were adapted to the normal diet and drinking for one week, set up a normal diet control group and DDC diet experimental group. The DDC model mice were fed with 0.1% DDC for 4 weeks and it mainly caused bile duct injury and intrahepatic cholestasis. After the modeling was completed, the mice in each group were anesthetized, shaved, and disinfected. All animal experiments were approved by the Animal Care and Use Committee of Shanghai University of Traditional Chinese Medicine (Shanghai, China) and were performed in accordance with the Ethical Guidelines for the Care and Use of Laboratory Animals.

2.3 HNK treatment

For the evaluation of HNK treatment on alleviation of DDC diet induced chronic cholestasis liver injury, we designed two experiment based on the chronic cholestasis liver injury model.

In the first experiment for the evaluation of the dosage-dependent hepatoprotective effect of HNK, four groups included a negative treated normal control group (NTC group), a negative treated DDC diet fed induced chronic cholestasis liver injury model group (DDC group), two dosages (2 mg/kg, and 10 mg/kg) of HNK treated chronic cholestasis liver injury group (HNK group). DDC diet was daily fed from day 1 to day 28 and HNK was daily intraperitoneal injected from day 15 to day 28. Mice were monitored daily and the body weight was recorded. After 4 weeks of DDC diet fed and responsible HNK treatment for 2 weeks, animals were anesthetized then with pentobarbital (50 mg/kg, intraperitoneal injection) and blood was collected via orbit. Then the abdominal cavity was opened, the liver was removed and weight. The liver was then rinsed with physiological saline. Left lobe and median lobes were separated and put into liquid nitrogen and stored at − 80 °C in ultra-low temperature refrigerator. Sections of the right lobe were fixed and stored in 4% paraformaldehyde overnight at 4 °C for paraffin sectioning.

In the second experiment for the analysis of hepatotoxicity and exploration of the inflammatory regulation of HNK treatment, four groups of mice included a negative treated normal control group (NTC), a high dosage of 10 mg/kg HNK treated normal control group (HNK), a DDC diet fed induced chronic cholestasis liver injury model group (DDC), and high dosage of 10 mg/kg HNK treated chronic cholestasis liver injury group (DDC + HNK). DDC diet was daily fed from day 1 to day 28 and HNK was daily intraperitoneal injected from day 15 to day 28. Mice were then monitored daily and the body weight were recorded. Then mice were anesthetized and EDTA treated blood was collected for flow cytometry analysis. After the liver was removed and weight, fresh livers were rinsed in PBS, the left lobe was used for single cell suspension preparation and flow cytometry analysis, median lobes were put into liquid nitrogen, and the right lobe was fixed in 4% paraformaldehyde for histopathological analysis.

2.4 Serum biochemistry

Blood samples were centrifuged to collect serum. Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were measured using an automatic biochemical analyzer with commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The concentrations of total BAs (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) in serum samples were analyzed by standard enzymatic assays using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Hepatic BAs were extracted from liver samples using 90% methanol and hepatic BAs were measured using a commercial assay kit (MAK309, Sigma–Aldrich, USA) as previously described.

2.5 Measurement of hepatic collagen content

Hepatic collagen deposition was determined biochemically by measuring the hydroxyproline content with modified method. In short, 500 mg liver samples underwent complete acid hydrolysis in 5 ml of 6 N hydrochloric acid solution at 110 °C for 16 h. After centrifugation at 2000 rpm for 5 min, 2 ml of supernatant was collected and neutralized with 8 N potassium hydroxide solution to pH 6–6.5 and was oxidized with 1 ml of 0.6 mol/l chloramine-T for 30 min. After oxidization, 1 ml of 7.5% p-dimethylaminobenzaldehyde was added and the mixture was incubated for 15 min at 65 °C. Absorbance was measured at 560 nm. Tissue content of hydroxyproline was determined using a standard curve generated from pure hydroxyproline. Results are expressed as μg/g of wet liver tissue. then hydroxyproline was quantitated photometrically as described.

2.6 Histopathology

The live tissue was fixed in 4% paraformaldehyde for at least 24 h and then was subjected to dehydration and penetration. The specimen was then embedded in paraffin. The section was cut at a thickness of 4 μm for haematoxylin and eosin (H&E) staining and Masson's trichrome staining. Pathological changes were observed by light microscopy.

2.7 Immunohistochemistry

The prepared sections (4 μm) were heated at 60 °C for 1 h and then rehydrated with xylene and different concentrations of alcohol. The sections were washed three times with phosphate buffer solution (PBS) to block non-specific binding sites, immersed in 5% bovine serum albumin (BSA) for 20 min at 37 °C and then incubated for 10 min. Specimens were incubated overnight at 4 °C with primary antibodies including CK19 (1:200), CD11b (1:200) and CD3 (1:100). On the second day, after incubation with an appropriate secondary antibody for 30 min at 37 °C, the sections were analysed with a diaminobenzidine kit to detect antibody binding. Finally, the slices were viewed on an ECLIPSE-Ci microscope (Nikon, Japan) and pictures were recorded using a digital camera Pannoramic MIDI II (3DHISTECH, Hungary) or Nano Zoomer S60 (HAMAMATSU, Japan).

2.8 BAs analysis

For serum and hepatic BAs measurements, The BAs concentration of all samples was measured by colorimetric analysis using commercial kit MAK309 (Sigma–Aldrich, USA) according to the manufacturer’s instructions. For the exaction of hepatic BAs, 200 mg of liver was homogenized in 1.0 ml of 70% ethanol, shaking incubated at 50 °C for 2 h and centrifuged at 15000 rpm for 15 min.

2.9 Analysis of individual bile acid composition by mass spectrometry

The profiles of individual BAs in biological samples were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS) referencing previous studies (Li et al., 2019, Xue et al., 2021). For sample preparation, a volume of 180 μl of ice-cold alkaline acetonitrile (5% NH₄OH in acetonitrile) was added to 20 μl of biological sample, vortexed, and centrifuged at 15000 rpm for 30 min at 4 °C. The supernatant was aspirated, evaporated under vacuum, and reconstituted in 40 μl of 50% acetonitrile. A volume of 10 μl supernatant was injected into the LC-MS/MS system for quantitative analysis. The instrument conditions were as follows: UHPLC- Q/Exactive mass spectrometer (Thermo Scientific, USA) with Column Hypersil Gold Dim (100 × 2.1 mm, 1.9 µm, USA). Mobile phase A was acetonitrile mobile and phase B was 5 mM ammonium formate with 0.1% formic acid in water. The flow rate was 0.3 ml/min and the gradient was set as, 0–2 min, 5% A–30% A; 2–5 min, 30% A–55% A; 5–16 min, 55% A–95% A; 16–18 min, 95% A; 18–20 min, 95% A–5% A. The mass spectrometer was equipped with an electrospray ionization source. All the BAs were detected in negative ion mode. Multiple reaction monitoring transitions of BAs and internal standard are listed in Supplementary Material of Table S1.

2.10 Quantitative real-time PCR

RNA from the liver was isolated using Trizol reagent (Invitrogen, USA) according to the manufacturers’ instructions. RNA concentrations were measured using NanoDrop 2000c spectrophotometer (Thermo Scientific, USA). Total liver RNA (2 μg) was reverse-transcribed into cDNA using Super Script II Reverse Transcriptase (Invitrogen, USA) and the oligo dT method. qPCR was performed using the Stepone Plus System (Applied Biosystems, USA) with SYBR green reagent (TaKaRaBio, Japan), according to manufacturers’ instructions. The ΔΔCT method was utilized to assess mRNA expression relative to that of GAPDH, which was used as an internal reference. The primers used for this study are listed in Supplementary Material Table S2.

2.11 Liver perfusion and cell separation

Fresh livers of left lobe were weight, then 500 mg liver tissue was perfused with 0.28 mg/mL collagenase IV (Sigma; C-5138) through the inferior vena cava. The dissociated cells were passed through a 70 mm cell strainer to remove clumps and undigested tissue. Cells were centrifuged mildly at 10 g for 5 min to pellet down hepatocytes and supernatant cells were collected by centrifugation at 350 g for 10 min. Cells were resuspended in 90 ml of running buffer (PBS pH 7.2, 0.5% BSA, and 2 mM EDTA) per 10⁷ cells, Erythrocytes were lysed with ACK Lysing Buffer (Thermofisher, USA). Remaining liver cells were suspended in the culture medium for Flow cytometry analysis.

2.12 Flow cytometry

Isolated single cell suspensions were prepared for flow cytometric analysis. Briefly, 5 million cells were incubated with a cocktail of fluorescent conjugated antibodies that summarized in Supplementary Material Table S3. Each incubation step was performed at 4 °C for 30 min and cells were subsequently washed two times in FACS buffer that PBS supplemented 1% fetal calf serum, 2.5 mM EDTA and 0.1% sodium azide. Prior to acquisition, labeled cells were incubated for 1 min with DAPI (Thermofisher, USA) at the final concentration of 1 mg/ml and washed with FACS buffer. Flow cytometric analysis was performed on a CytoFLEX flow cytometer (Beckman Coulter, USA) and analyzed using FlowJo software (TreeStar, Olten, Switzerland). Debris was excluded based on physical parameters (forward and side scatter characteristics, respectively).

2.13 Statistical analysis

All data were collected and analyzed with GraphPad Prism Software version 9.0 for Windows (GraphPad, USA). The data are presented graphically as mean values ± standard deviation (SD). Data significance was compared by the one-way or two-way ANOVA method, followed by Tukey's multiple comparisons test. Statistically significant differences were marked as values of *p < 0.05, **p < 0.01, ***p < 0 0.001, and ***p < 0 0.0001.

3.1 HNK attenuated DDC-induced ductular proliferation

We first established a chronic cholestasis liver injury model. DDC feed mice were reported as a successful chronic cholestasis liver injury model in a dose- and time-dependent manner (Pose et al., 2019, Li et al., 2021). In most cases, primary damage to the biliary epithelium leads to the development of cholestasis (Beretta-Piccoli et al., 2019). Long-standing cholestasis causes an ordered bile duct proliferation or typical ductular reaction (Mariotti et al., 2019). In our experiment, a 0.1% DDC containing diet was feed into C57BL/6J mice for 4 weeks showed the hepatomegaly and body weight loss. The significant decreasing body weight and the increasing liver weight were accompanied by the increasing of liver/body weight ratio from 6.1% to 12.9% (Fig. 1A). Macroscopic analysis of the livers of DDC feed mice showed a brownish colour change and hepatomegaly, indicating cholestasis and liver injury. In line with these macroscopic results, the serological analysis also revealed liver injury and cholestasis (Fig. 1B), as shown by significantly increased levels of AST, ALT, and ALP. To evaluate chronic cholestatic liver injury changes of DDC diet, we performed a pathological analysis of livers. The histopathology results of HE staining, masson staining and CK19 immunochemistry of the liver sections showed the typical ductular reaction (Fig. 1C). The ductular reaction was reported to emerge and originate from proliferation of pre-existing bile duct epithelial cells(Mariotti et al., 2019, Pose et al., 2019). Moreover, DDC induced obvious hepatic concentric periductal fibrosis (a typical feature of human cholangitis) that is characterized as chronic obstruction of the biliary tree, accumulation of extracellular matrix at portal tracts, surrounding newly formed bile ducts.

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Fig. 1

HNK attenuated DDC-induced ductular proliferation. According to the description in the materials and methods, C57BL/6J mice were fed with DDC diet or treated with different dosages of HNK. (A) The body weight, liver weight, and liver weight to body weight percentage in the DDC diet and HNK treatment groups. (B) The serum levels of ALT, AST, and ALP in the DDC diet and HNK treatment groups. (C) Representative images of hepatic HE staining, Masson staining and immunohistochemistry for CK19 were shown in the DDC diet and HNK treatment groups. The data were presented graphically as mean ± SD and compared by the one-way ANOVA method, followed by Tukey's multiple comparisons test. Five mice for each group from the first in vivo experiment. Significant differences were marked as *p < 0.05, **p < 0.01, ***p < 0 0.001, and ****p < 0 0.0001.

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To evaluate the hepatoprotective effect of HNK after referencing the previous studies(Sulakhiya et al., 2015, Elfeky et al., 2020), we used two dosages of HNK (2 mg/kg/day and 10 mg/kg/day) for the treatment of chronic cholestasis liver injury. It was found the hepatomegaly and body weight loss was reversed by the HNK treatment in a dose dependent manner, as well as the elevation of liver/body weight ratio. Moreover, the increased levels of serological ALT, AST and ALP were relieved. The pathological analysis of livers revealed the hepatoprotective effect of HNK even though typical ductular reaction, fibrosis and infiltration of inflammatory cells around the portal area were alleviated in the two dosages of HNK treatment groups.

3.2 HNK attenuated DDC-induced BAs accumulation

DDC diet feed in mice often induced hepatic accumulation of bilirubin and BAs concentration(Li et al., 2021). The significant elevation of serum total bilirubin and direct bilirubin were observed in DDC diet feed mice with chronic cholestasis liver injury (Fig. 2A). HNK alleviated the elevation of serum total bilirubin and direct bilirubin. Moreover, DDC induced significant increasing of serum and hepatic BAs levels. HNK treatment resulted in dosage-dependent decreasing of serum and hepatic BAs accumulation (Fig. 2B). BAs included a large family of molecules with steroidal structure that synthesized from cholesterol in the liver(Matz-Soja 2020). BAs are physiological detergents that facilitate excretion, absorption, and transport of fats and sterols in the liver. Recent studies showed they modulate bile flow, lipid secretion, key enzymes that involved in overall metabolic homeostasis(Fuchs and Trauner 2022). However, BAs have potent toxic properties for initiating and perpetuating bile duct injury(Ahmadi et al., 2021, Dotan et al., 2022), we then measured primary and secondary BAs. It was found DDC induced CA, β-MCA, TCA, T-α-MCA, T-β-MCA and TCDCA significant increase in hepatic tissue (Fig. 2C). 2 mg/kg HNK countered the elevation of TCA, T-α-MCA, and TCDCA, while 10 mg/kg HNK reversed the elevation of β-MCA, TCA, T-α-MCA, T-β-MCA and TCDCA. Furthermore, the secondary BAs, such as ω-MCA, TDCA, TUDCA and T-ω-MCA were also increased after DDC feed for 4 weeks, and HNK countered all the four elevation secondary BAs (Fig. 2D). These results indicated HNK attenuated DDC induced hepatic BAs accumulation and alleviated BAs mediated cytotoxicity in the liver.

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Fig. 2

HNK attenuated DDC-induced bile acids accumulation. (A) The serum total bilirubin and direct bilirubin concentrations in the DDC diet and HNK treatment groups. (B) The serum bile acid and hepatic bile acid levels in the DDC diet and HNK treatment groups. (C) The hepatic individual primary bile acid levels in the DDC diet and HNK treatment groups. (D)The hepatic individual secondary bile acid levels in the DDC diet and HNK treatment groups. (E) The hepatic gene expression of Ck19 in the DDC diet and HNK treatment groups. (F) The hepatic gene expression of Cyp7a1 and Cyp8b1 in the DDC diet and HNK treatment groups. (G) The hepatic gene expression of Cyp27a1 and Cyp7b1 in the DDC diet and HNK treatment groups. The data were presented graphically as mean ± SD and compared by the one-way or two-way ANOVA method, followed by Tukey's multiple comparisons test. Five mice for each group from the first in vivo experiment. Significant differences were marked as *p < 0.05, **p < 0.01, ***p < 0 0.001, and ****p < 0 0.0001.

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The hepatotoxicity of DDC feed mice were also reflected by the significant increasing of hepatic CK19 gene expression (Fig. 2E). It was lined with the liver immunohistochemical results of CK19. HNK treatment resulted in decreasing expression of hepatic CK19 gene expression. Based on the toxic BAs hypothesis (Woolbright and Jaeschke 2019), we further measured the gene expression of enzyme responsible for BAs synthesis. It was found the two enzymes of Cyp7a1 and Cyp8b1, were significant decrease after DDC feed, as previous study(Wen et al., 2021) (Fig. 2F). These enzymes were involved in the classical pathway of BAs synthesis(Fuchs and Trauner 2022). HNK alleviated the inhibition of these two enzymes and implied HNK reprogramed the synthesis of hepatic BAs. Moreover, the alternative pathway of BAs synthesis genes Cyp27a1 and Cyp7b1 were inhibited by DDC diet feed (Fig. 2G). HNK showed dosage-dependent increase of Cyp27a1 and Cyp7b1 genes expression found a significant reduction of BAs concentration after HNK treatment. indicating HNK was partly beneficial to the excretion of BAs and maintenance of BAs homeostasis. The relationships between the hepatobiliary-protective effect of HNK and BAs regulation was described for the first time. It was reported that HNK can evidently relieve hepatocyte injuries and help to promote the secretion of BAs and benefit the jaundice.

3.3 HNK alleviated DDC-induced BAs metabolism, uptake, efflux, and transporter

Since hepatic BAs were elevated and these BAs had potential toxic properties of membrane disruption(Woolbright and Jaeschke 2019). At present, novel therapeutic approaches for the treatment of cholestatic liver diseases focused on the metabolism and circulation of BAs(Fuchs and Trauner 2022). We then investigated the effect of HNK on the BAs regulation, including metabolism, efflux, intake and circulation. It was consistent with previous research results (Fickert et al., 2007)that DDC induced gene expression elevation of phase I metabolism enzyme of Cyp3a11 and Cyp2b10 (Fig. 3A), as well as phase II metabolism enzymes of Sult2a1 and Ugt1a1 (Fig. 3B). These activation of BAs metabolism was mainly resulted in the high BAs. HNK treatment groups showed moderate arise of the genes expression that were responsible for BAs phase I and II metabolism. BAs recirculate through the liver, bile ducts, small intestine and portal vein to form an enterohepatic circuit (St-Pierre et al., 2001). DDC induced decreasing genes expression of Ntcp and Oatp1(Fig. 3C). These two genes responsible for the uptake of BAs into hepatocytes. The high levels of hepatic BAs impaired the uptake of BAs into hepatocytes. The BAs efflux into bile were also blocked since the genes expression of Bsep and Mrp2 was decreasing (Fig. 3D). It was interesting HNK treatment alleviated BAs uptake into hepatocytes and efflux into bile. High dosage of 10 mg/kg HNK showed a significant stronger activation of Ntcp, Oatp1, Bsep and Mrp2 than low dosage of 2 HNK. The accumulated hepatic BAs efflux into systemic circulation through canalicular export pumps of Osta, Ostb, Mrp3 and Mrp4, thus resulted in the accumulation of BAs in the serum. Our results showed DDC diet induced decreasing expression of Osta, Ostb, and Mrp3 (Fig. 3E). HNK treatment encountered the BAs alternative basolateral efflux pumps. Taken together, HNK partially recovered the BAs transporters and the bile flow was consistent with the relatively moderate cholestasis liver injury.

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Fig. 3

HNK alleviated DDC-induced bile acids metabolism, uptake, efflux, and transporter. (A) The hepatic gene expression of Cyp3a11 and Cyp2b10 in the DDC diet and HNK treatment groups. (B) The hepatic gene expression of Sult2a1 and Ugt1a1 in the DDC diet and HNK treatment groups. (C) The hepatic gene expression of Ntcp and Oatp1 in the DDC diet and HNK treatment groups. (D) The hepatic gene expression of Bsep and Mrp2 in the DDC diet and HNK treatment groups. (E) The hepatic gene expression of Osta, Ostb, Mrp3 and Mrp4 in the DDC diet and HNK treatment groups. The data were presented graphically as mean ± SD and compared by the one-way ANOVA method, followed by Tukey's multiple comparisons test. Five mice for each group from the first in vivo experiment. Significant differences were marked as *p < 0.05, **p < 0.01, ***p < 0 0.001, and ****p < 0 0.0001.

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3.4 HNK attenuated DDC-induced inflammatory response

Previous HE staining results showed obvious infiltration of inflammatory cells surrounding newly formed bile ducts (Fig. 1C). We thus performed immunohistochemically analysis of liver infiltrating inflammatory cells based on staining of CD11b and CD3. CD11b was mainly expressed on the surface of myeloid cells including monocytes, macrophages and Kupffer cells. Functionally, CD11b regulates myeloid cells adhesion and migration to the injured live and mediate the inflammatory response for the hepatoprotective effect. We found a diffuse high expression of CD11b positive cells in the liver of DDC feed mice (Fig. 4A). HNK treatment resulted in moderate infiltration of myeloid cells in the liver. CD3 positive lymphocytes were also aggregated and surrounded the newly formed bile ducts. HNK treatment group showed a decreasing infiltration of CD3 positive cells in the DDC induced cholestasis-related liver. Mcp-1 was an important chemokine that regulates migration and infiltration of monocytes and macrophages. Previous studies also showed the increasing of Mcp-1 in the DDC induced cholestasis-related liver(Wen et al., 2021). We verified the increased Mcp-1 gene expression in the DDC group (Fig. 4B). The descending Mcp-1 expression in the HNK treatment groups indicated its inhibition of infiltration of inflammatory cells in the cholestasis-related liver. The cytokines of Tnf-a, Il-1β, and Il-6 were not only associated with inflammatory cells, but also played multiple effects on ductular reactions and fibrosis(Pose et al., 2019). We then measured the gene expression of inflammatory related cytokines in the liver. DDC diet feed group showed abnormally elevation of Tnf-α, Il-1β, and Il-6 (Fig. 4C). HNK treatment alleviated the elevation of classical cytokines of Tnf-a, Il-1β, and Il-6. During this inflammatory response, inflammatory cells also release TGF-β1 to enhance their protein synthesis and metabolic activity(Gough et al., 2021). Among the pro-inflammatory and pro-fibrotic properties, Tgfb1 stimulate recruitment, proliferation and activation of a various of cells, especially fibroblasts for the deposition of extracellular matrix and stabilize the fibrotic tissue architecture. We confirmed the elevation of Tgfb1 gene expression in the DDC feed group(Wen et al., 2021) (Fig. 4D). HNK treatment showed a dosage-dependent alleviation of elevating Tgfb1 and indicated the consistent inhibition of fibrosis phenotypes.

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Fig. 4

HNK attenuated DDC-induced inflammatory response. (A) Representative images of hepatic HE staining and immunohistochemistry for CK19, CD11b and CD3 were shown in the DDC diet and HNK treatment groups. (B) The hepatic gene expression of Mcp-1 in the DDC diet and HNK treatment groups. (C) The hepatic gene expression of Tnfa, Il6, and Il1b in the DDC diet and HNK treatment groups. (D) The hepatic gene expression of Tgfb1 in the DDC diet and HNK treatment groups. The data were presented graphically as mean ± SD and compared by the one-way ANOVA method, followed by Tukey's multiple comparisons test. Five mice for each group from the second in vivo experiment. Significant differences were marked as *p < 0.05, **p < 0.01, ***p < 0 0.001, and ****p < 0 0.0001.

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3.5 HNK alleviated DDC-induced infiltration of inflammatory cells

Previous studies revealed the decreasing of Kupffer cells and increasing of monocyte derived macrophages in the DDC induced cholestasis-related liver(Gough et al., 2021). Lymphocytes were also investigated for the development of DDC induced cholestasis liver injury(Fickert et al., 2007). To analysis the infiltration of inflammatory cells and reveal the hepatotoxicity of high dosage of HNK, we designed dependent experiment of high dosage of HNK (10 mg/kg/day) for 28 days continuously treatment for the control group and DDC diet feed groups. It was consistent with previous study that DDC induced hepatomegaly and weight loss were reversed by HNK treatment (Fig. 5A). It was noted HNK treatment in control mice showed no significant difference of body weight and liver weight. The peripheral blood mononuclear cells and hepatic single cells suspension were prepared for flow cytometry. An optimized flow cytometry strategy for the simultaneous analysis of hepatic lymphoid cells and myeloid cells were developed based on literature(Liu et al., 2020, Cossarizza et al., 2021) (Fig. 5B). It was found there was a significant increase of leukocytes based on liver CD45 + cells in the DDC induced cholestasis-related liver (Fig. 5C). HNK treatment group showed a descending infiltration of leukocytes when compared with DDC diet induced cholestasis liver injury groups. However, there was no significant difference of infiltrating leukocytes when compared HNK treated control mice when compared with NTC group. For the analysis of myeloid cells of CD11b + cells, we further separated monocyte derived macrophages and kupffer cells. It was found DDC induced increasing infiltration of monocyte derived macrophages (MoDMacs) (Fig. 5D). However, the Kupffer cells was depleted after DDC diet feed. HNK treatment in DDC diet feed mice not only increased the infiltrated MoDMacs percentage, but also preserved the depletion of Kupffer cells when compared with DDC diet feed mice. For the lymphoid cells, we first distinct CD3 + lymphocytes, and found there was a significant increasing of infiltrating CD3 + T lymphocytes after DDC feed, and a moderate decline of CD3 + T lymphocytes infiltration after HNK treatment (Fig. 5E). Moreover, CD4 + and CD8 + T lymphocytes were analyzed and we confirmed the significant increasing infiltration of CD8 + T lymphocytes and decreasing infiltration of CD8 + T lymphocytes after DDC diet feed. HNK treatment showed a decreasing of CD8 + T lymphocytes and an alleviation of CD4 + T lymphocytes infiltration in the DDC induced cholestasis-related liver. The infiltration of leukocytes was associated with production and secretion of pro-inflammatory cytokines. Thus we measured the hepatic cytokines of TNF-α, and IL-6 (Fig. 5F). There was significant increasing of cytokines TNF-αand IL-6 after DDC feed, and moderate decreasing of cytokines TNF-α and IL-6 after HNK treatment. These result indicated the DDC induced infiltration of inflammatory cells and production of cytokines were countered by the HNK treatment.

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Fig. 5

HNK alleviated DDC-induced infiltration of inflammatory cells. (A) The body weight, liver weight, and liver weight to body weight percentage in the DDC diet and HNK treatment groups. (B) The representative flow cytometry strategy for analyzing the hepatic infiltrating immunocytes. (C) The hepatic leukocyte percentage in the DDC diet and HNK treatment groups. (D) The hepatic MoDMacs and Kupffer cells percentages in the DDC diet and HNK treatment groups. (E) The hepatic CD3 + T cells in lymphoid cells, as well as CD4 + and CD8 + T cells in CD3 + T cells percentages in the DDC diet and HNK treatment groups. (F) The hepatic inflammatory levels of TNF-α and IL-6 in the DDC diet and HNK treatment groups. The data were presented graphically as mean ± SD and compared by the one-way ANOVA method, followed by Tukey's multiple comparisons test. Five mice for each group from the second in vivo experiment. Significant differences were marked as *p < 0.05, **p < 0.01, ***p < 0 0.001, and ****p < 0 0.0001.

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