Mice

This is an in vivo and in vitro study performed on mice. C57BL male mice, aged 6–8 weeks and weighing 20–25 g, were selected for in vitro and in vivo experiments. All animal experiments were conducted in strict compliance with the ARRIVE guidelines¹⁹. The National Research Council’s Guide for the Care and Use of Laboratory Animals was also followed to guarantee the appropriate care and ethical treatment of the animals involved.

Cell culture and transfection

Mouse pancreatic stellate cells (mPSCs) were isolated and cultured in complete DMEM/F12 medium (Gibco). The experiments in this study utilized mPSCs at passages 1–2, which were then stimulated with nicotine (Sigma Aldrich). In addition, mPSCs were transfected with small interfering RNA (siRNA) obtained from Syngentech (China) using Lipofectamine RNAiMAX (Invitrogen).

Cell proliferation

Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, Lablead, China). A total of 2.0×10³ mPSCs were seeded in each well of a 96-well plate. The absorbance at 450 nm was measured to quantify cell proliferation.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from mPSCs and pancreatic tissues using TRIzol reagent (Invitrogen). Reverse transcription was performed using the PrimeScript RT Master Mix (Takara). qRT-PCR was conducted on an ABI 7500 system (Applied Biosystems) using the SYBR Green Master Mix and primers listed in Supplementary file Table 1. The relative mRNA levels were determined using the Livak (2-ΔΔCt) method.

Assessment of mitochondrial reactive oxygen species (ROS)

A total of 2.0×10³ mPSCs were seeded in each well of a 96-well plate. Cells were incubated in 500 nM MitoSOX Red (Invitrogen) for 30 min and Hoechst 33342 (Beyotime Biotechnology, China) for 10 min, protected from light. Fluorescence intensity of Hoechst (ex/em: 350/461 nm) and MitoSOX (ex/em: 396/610 nm) were detected, respectively. The intensity of MitoSOX was normalized to that of Hoechst.

Measurement of malondialdehyde (MDA)

MDA levels in mPSCs and serum were quantified using the MDA assay kit (NJJCBIO, China). After adding the relevant reagents and incubating at 95^oC for 40 min, the absorbance at 530 nm was determined.

RNA sequencing (RNA-seq)

RNA-seq was performed by Novogene Co., Ltd. (Beijing, China). Briefly, total RNA was extracted from mPSCs using TRIzol reagent (Invitrogen). Qualified libraries were sequenced on the Illumina NovaSeq 6000 platform. Clean reads were aligned to the reference genome using Hisat2 (v2.0.5). The gene expression levels were estimated by calculating the expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced (FPKM) for each gene. We utilize the Gene Set Enrichment Analysis (GSEA) analysis tool available at http://www.broadinstitute.org/gsea/index.jsp for conducting GSEA.

Assessment of mitochondrial Ca2+ levels

Cells were treated with 4 μM Rhod-2 AM (Invitrogen) for 20 min and Hoechst 33342 (Beyotime) for 10 min, both in a light-protected environment. Subsequently, fluorescence microscopy was employed to visualize the signals. The intensity of Rhod-2 was normalized to that of Hoechst.

Western blotting analysis

Protein extraction from mPSCs and pancreatic tissues was lysed using RIPA buffer (Solarbio) supplemented with protease and phosphatase inhibitors. The protein concentration was determined using the BCA Assay Kit (Beyotime). Protein samples underwent SDS-PAGE electrophoresis and were transferred onto PVDF membranes. Subsequently, the membranes were incubated overnight at 4^oC with the primary antibody, followed by a 1-h incubation with the secondary antibody. Details of the antibodies used are provided in Supplementary file Table S2.

Assessment of mitochondrial membrane potential (MMP)

MMP was assessed using the Mito Probe JC-1 assay kit (Invitrogen). Cells were incubated with 2 μM JC-1 for 15–30 min. The red and green signals of JC-1 were visualized using a fluorescence microscope. The ratio of red/green fluorescence intensity was calculated as a measure of MMP.

CP model and treatment

The CP model was established by repeated intraperitoneal injections of cerulein, 50 μg/kg, six times per day with a 1-hour interval, three days a week for four weeks²⁰. Nicotine was administered via intragastric administration at a dose of 6 mg/kg/day, four times a day for four weeks^6,21. Ru360 was administered by intraperitoneal injection, 240 μg/kg, one hour before nicotine, once a day for four weeks²².

Histology

Pancreas tissues were fixed in 4% paraformaldehyde. The degree of pancreatic fibrosis was assessed using hematoxylin and eosin (H&E), Masson trichrome, and Sirius red staining performed by Servicebio technology.

Immunohistochemical staining

Immunohistochemical staining of α-SMA in pancreatic tissue was performed to assess the activation of mPSCs. The slides were first incubated with α-SMA antibody (Abcam, #ab5694) overnight at 4°C. Subsequently, the slides were incubated with horseradish peroxidase-conjugated secondary antibodies (ZSGB-BIO) for 20 min.

Statistical analysis

The results are presented as mean ± standard deviation (SD). Statistical analysis was performed using the unpaired t-test or one-way ANOVA with SPSS software (version 24.0, Chicago, USA). A p<0.05 was considered statistically significant.

Nicotine promotes activation and induces mitochondrial oxidative stress in mPSCs

In our previous study, we discovered that nicotine has the ability to enhance the activation of hPSCs and induce mitochondrial oxidative stress. The most significant impact was observed when cells were treated with 1μM nicotine for 48 hours⁷. In this study, we extracted and cultured mPSCs in vitro to simulate the condition of PSCs in CP. We found that nicotine treatment (1 μM, 48 h) significantly promoted the activation of mPSCs, as evidenced by a notable increase in cell proliferation (p<0.05) (Figure 1A) and an upregulation in the mRNA expression level of α-SMA, a marker of PSCs activation (p<0.01) (Figure 1B). Additionally, nicotine also stimulated fibroblast activity in mPSCs and elevated the mRNA expression level of Type I collagen (Col I), the primary constituent of the extracellular matrix (ECM), suggesting that nicotine may disrupt ECM metabolism in mPSCs (p<0.01) (Figure 1C).

Figure 1

Nicotine promotes activation and induces mitochondrial oxidative stress in mouse pancreatic stellate cells (mPSCs): A) Relative proliferation and viability of mPSCs determined by CCK-8 assays; B, C) mRNA levels of α-SMA and Col1 in mPSCs; D) Mitochondrial reactive oxygen species (ROS) levels in mPSCs; E) Intracellular malondialdehyde (MDA) levels in mPSCs

Furthermore, nicotine aggravated mitochondrial oxidative stress in mPSCs. The levels of mitochondrial ROS in the nicotine group were significantly elevated compared to the negative control group (p<0.05) (Figure 1D). Moreover, the level of malondialdehyde (MDA), a product of lipid peroxidation, was significantly increased (p<0.01) (Figure 1E). These findings indicate that nicotine may induce damage to mPSCs through oxidative stress.

The potential involvement of the calcium signaling pathway in the promotion of mPSCs activation by nicotine

To investigate the underlying mechanism by which nicotine promotes mPSCs activation, transcriptome sequencing was conducted comparing the nicotine-treated group with the negative control group of mPSCs. Principal component analysis (PCA) revealed a significant distinction between the two groups (Supplementary file Figure S1A). GSEA indicated an upregulation of the calcium signaling pathway in the nicotine treatment group compared to the control group, suggesting that nicotine may exert its effects through this pathway (NES=1.272, p=0.046) (Supplementary file Figure S1B). Consequently, we examined mitochondrial Ca²⁺ levels in nicotine-treated mPSCs using Rhod-2 probes under fluorescence microscopy (Figure 2A). Notably, the mitochondrial Ca²⁺ level in mPSCs exhibited a significant increase upon nicotine stimulation (p<0.001) (Figure 2B), implying a potential link between nicotine-induced biological changes in mPSCs and alterations in mitochondrial calcium homeostasis.

Figure 2

The potential involvement of the calcium signaling pathway in the promotion of mPSCs activation by nicotine: A) Fluorescent microscopy images showing Rhod-2 staining of mitochondrial Ca²⁺ and Hoechst staining of the cell nucleus in mPSCs (scale bars=100 μm); B) Measurement of mitochondrial Ca²⁺ levels by normalizing the fluorescence intensity of Rhod-2 to that of Hoechst in mPSCs; C) mRNA levels of mitochondrial calcium uniporter (MCU) in mPSCs; D) Protein expression levels of α-SMA and MCU

Considering the association of nicotine’s effects on mPSCs with mitochondrial Ca²⁺ levels, we further investigated the impact of nicotine on proteins involved in mitochondrial calcium transport. Among these proteins, the mitochondrial calcium uniporter (MCU) plays a crucial role in facilitating mitochondrial calcium influx. Interestingly, our findings demonstrated a substantial elevation in MCU mRNA levels following nicotine stimulation (p<0.05) (Figure 2C), accompanied by an increasing trend in MCU protein expression (Figure 2D). These results suggest that nicotine modulates mitochondrial calcium homeostasis, potentially mediated by upregulating MCU, thus contributing to mPSCs activation.

Nicotine aggravates mPSCs activation through the dysregulation of mitochondrial calcium homeostasis and oxidative stress mediated by MCU

Based on the preliminary findings, we employed siRNA transfection to knock down MCU in mPSCs to investigate its impact on nicotine-induced mPSCs activation and oxidative stress. The cells were divided into four groups: control group (siCtrl), nicotine group (siCtrl+Nic), MCU knockdown group (siMCU), and MCU knockdown + nicotine group (siMCU+Nic). Compared to the nicotine group, the application of nicotine after MCU knockdown resulted in a significant decrease in mitochondrial Ca²⁺ levels, whereas the sole knockdown of MCU did not exhibit significant changes in mitochondrial Ca²⁺ levels compared to the control group (p<0.01) (Figure 3A). Additionally, we investigated the impact of MCU on mitochondrial oxidative stress. MCU knockdown markedly attenuated the nicotine-induced elevation of mitochondrial ROS levels and intracellular MDA levels (p<0.05) (Figures 3B and 3C). The enhanced activation of mPSCs by nicotine was also suppressed by MCU knockdown. Cell proliferation, which was significantly increased after nicotine treatment (p<0.01), was reversed by MCU knockdown (Figure 3D). MCU knockdown effectively reversed the changes in mRNA levels of α-SMA and collagen I induced by nicotine (p<0.05) (Figures 3E and 3F) and alleviated the elevation of α-SMA protein levels (Supplementary file Figure S2A).

Figure 3

Nicotine aggravates mPSCs activation through the dysregulation of mitochondrial calcium homeostasis and oxidative stress mediated by MCU: A) Measurement of mitochondrial Ca²⁺ levels by normalizing the fluorescence intensity of Rhod-2 to that of Hoechst in mPSCs; B) Mitochondrial ROS levels in mPSCs; C) Intracellular MDA levels in mPSCs; D) Relative proliferation and viability of mPSCs determined by CCK-8 assays; E, F) mRNA levels of α-SMA and Col1 in mPSCs

In addition, we investigated the effects of nicotine on mitochondrial dynamics-related proteins. Our results demonstrated that MCU knockdown significantly reduced the elevated mRNA levels of DRP1 in mPSCs stimulated by nicotine (p<0.0001) (Supplementary file Figure S2B). Furthermore, we utilized JC-1 to assess MMP and observed under a fluorescence microscope. JC-1 exhibited potential-dependent accumulation in mitochondria, and mitochondrial depolarization was indicated by a decrease in the red/green fluorescence intensity ratio in the aggregate form/monomer form. MCU knockdown alleviated the nicotine-induced reduction of MMP in mPSCs (Supplementary file Figure S2C).

Combining these findings with our previous research, we propose that nicotine may upregulate MCU, leading to increased mitochondrial calcium levels, exacerbation of mitochondrial oxidative stress, enhanced mitochondrial fission, and decreased MMP, ultimately promoting mPSCs activation.

The MCU inhibitor Ru360 alleviates nicotine-aggravated pancreatic fibrosis in CP mice

Based on in vitro experiments, we further validated our findings in an in vivo experiment. We investigated the impact of nicotine on the severity of pancreatic fibrosis in CP mice and explored the therapeutic effects of the MCU inhibitor Ru360. We established a CP mouse model by intraperitoneal injection of cerulein and divided the CP mice into three groups: control group (Cae), nicotine group (Cae+Nic), and Ru360 group (Cae+Nic+Ru360) (Supplementary file Figure S3A). After sample collection, multiple parameters were analyzed, including body weight, pancreas weight, extent of pancreatic injury and fibrosis, activation of PSCs, and oxidative stress levels in pancreatic tissue.

The results demonstrated that nicotine significantly decreased the pancreas weight-to-body weight ratio in CP mice, which was effectively alleviated by the administration of Ru360 (p<0.05) (Supplementary file Figure S3B). Additionally, nicotine-induced histological changes in the pancreatic tissue, such as glandular atrophy and fibrosis, were observed (Figure 4A). Quantitative analysis of pancreatic fibrosis was conducted by measuring the positive area in Masson-trichrome staining, demonstrates that Ru360 significantly attenuated the exacerbated pancreatic fibrosis induced by nicotine (p<0.001) (Figure 4B). Furthermore, nicotine-induced upregulation of Col1 mRNA levels was significantly reversed by Ru360 (p<0.0001) (Figure 4C).

Figure 4

The MCU inhibitor Ru360 alleviates nicotine-aggravated pancreatic fibrosis in CP mice: A) Histological analysis of pancreatic tissues using H&E, Masson Trichrome, Sirus Red, and Sirus Red(polarizing) staining, along with immunohistochemical staining of α-SMA (scale bars=100 μm); B) Quantitative evaluation of the pancreatic fibrosis; C) mRNA levels of Col1 in pancreatic tissues; D) Immunohistochemical staining of α-SMA in pancreatic tissues; E) mRNA levels of α-SMA in pancreatic tissues; F) Measurement of serum MDA levels

Moreover, Ru360 significantly inhibited the promoting effect of nicotine on PSCs activation. It effectively reversed the increased expression of α-SMA in pancreatic tissue induced by nicotine (p<0.05) (Figure 4D). This reversal was also evident at mRNA expression levels (p<0.05) (Figure 4E). Similarly, the serum levels of MDA were significantly elevated in the nicotine group, while the Ru360 group exhibited a significant reduction in MDA content (p<0.0001) (Figure 4F), indicating that Ru360 could reverse nicotine-aggravated oxidative stress damage in the pancreas of CP mice.

In summary, Ru360 effectively inhibits the nicotine-induced upregulation of MCU expression, thereby ameliorating oxidative stress damage in PSCs, ultimately alleviating the activation of PSCs and pancreatic fibrosis in CP mice exacerbated by nicotine.

Limitations

This study has certain limitations. Firstly, the in vivo experiments did not involve the use of MCU gene knockout mice, and only one mitochondrial calcium-specific inhibitor was applied, which may limit the persuasiveness of the research findings. Additionally, we did not investigate the reciprocal interactions between intracellular Ca²⁺ and mitochondrial Ca²⁺. Therefore, further research is still needed to explore the underlying mechanisms of nicotine in CP progression.