Chang'an decoction (肠安方) alleviates endoplasmic reticulum stress by regulating mitofusin 2 to improve colitis

doi:10.19852/j.cnki.jtcm.20240308.001

Abstract

Abstract:

OBJECTIVE: To evaluate the protective effects of Chang'an decoction (肠安方, CAD) on colitis, and investigate the potential mechanisms underlying these effects from the perspectives of endoplasmic reticulum (ER) stress induced by mitofusin 2 (MFN2).

METHODS: The composition of CAD was identified by liquid chromatography-mass spectrometry technology. A mice model of dextran sulfate sodium (DSS) induced colitis was established and therapeutic effects of CAD were determined by detecting body weight, disease activity index, colon length and histopathological changes. Then, the expression levels of MFN2, ER stress markers and Nucleotide-binding domain and leucine-rich repeat protein3 (NLRP3) relevant proteins were detected by polymerase chain reaction (PCR), Western blot, immunohistochemistry and immunofluorescence staining. Subsequently, knockdown and overexpression cell model were constructed to further investigate the underlying mechanism of MFN2 mediating ER stress and energy metabolism by PCR, Western blot, electron microscopy and reactive oxygen species (ROS) staining. Finally, inflammatory indicator and tight junction proteins were measured by PCR and immunofluorescence staining to evaluate the protective effects of CAD.

RESULTS: Results showed that the indispensable regulatory role of MFN2 in mediating ER stress and mitochondrial damage was involved in the protective effects of CAD on colitis in mice fed with DSS. Network pharmacology analysis also revealed CAD may play a protective effect on colitis by affecting mitochondrial function. In addition, our data also suggested a causative role for MFN2 in the development of inflammatory responses and energy metabolic alterations by constructing a knockdown and overexpression cell model whereby alter proper ER-mitochondria interaction in Caco-2 cells. Furthermore, relative expression analyses of ER stress markers and NLRP3 inflammasome showed the onset of ER stress and activation of NLRP3 inflammasome, which is consistent with the above findings. In contrast, intervention of CAD could improve the mucosal barrier integrity and colonic inflammatory response effectively through inhibiting ER stress response mediated by MFN2.

CONCLUSION: CAD could alleviate ER stress by regulating MFN2 to exert therapeutic effects on DSS-induced colitis, which might provide an effective natural therapeutic approach for the treatment of ulcerative colitis.

Key words: colitis, mitofusin 2, endoplasmic reticulum stress, mitochondrial damage, Chang'an decoction

CHEN Youlan, DING Mingming, HUANG Chaoyuan, ZHENG Yiyuan, LIU Fengbin. Chang'an decoction (肠安方) alleviates endoplasmic reticulum stress by regulating mitofusin 2 to improve colitis[J]. Journal of Traditional Chinese Medicine, 2024, 44(3): 427-436.

Figures/Tables 4

Figure 1 CAD alleviated DSS-induced colitis in mice A: animal experimental flow; B: body weight change; C: DAI scores; D: length of colons; E: statistical graph of colon length; F: HE staining in colon tissues, Scale bars: 100 μm; F1: CON; F2: DSS; F3: CAD-L; F4: CAD-M; F5: CAD-H. CON: control group; DSS: ulcerative coloits group; CAD-L: low-dose Chang'an decoction (0.5 g/mL); CAD-M: medium-dose Chang'an decoction (1 g/mL); CAD-H: high-dose Chang'an decoction (2 g/mL). The drug was administered by gavage once daily for 6 d. CAD: Chang'an decoction; DAI: disease activity index; DSS: dextran sulfate sodium; HE: hematoxylin and eosin. Results are presented as mean ± standard deviation. aP < 0.001 vs CON; bP < 0.001 vs DSS, n = 3-6.

Figure 2 Effects of CAD on ER stress and NLRP3 inflammasome activation in mice colon tissue A: Western blot analyses of GRP78, p-PERK, PERK, p-eIF2α and eIF2α in colon tissues; B: statistical graphs of GRP78/β-actin; C: statistical graphs of pPERK/PERK; D: statistical graphs of p-eIF2α/eIF2α; E: IHC staining of GRP78 in colon tissues; F: statistical graphs of GRP78 expression in IHC; Scale bars: 50 μm; E1: CON; E2: DSS; E3: CAD-L; E4: CAD-H. G: Western blot analyses of NLRP3 and ASC in colon tissues. H: The statistical graphs of NLRP3/β-actin. I: statistical graphs of ASC/β-actin; J: IHC staining of ASC in colon tissues; J1: CON; J2: DSS; J3: CAD-L; J4: CAD-H. K: statistical graph of ASC expression in IHC; Scale bars: 50 μm; CON: control group; DSS: ulcerative coloits group; CAD-L: low-dose Chang'an decoction (0.5 g/mL); CAD-H: high-dose Chang'an decoction (2 g/mL). The drug was administered by gavage once daily for 6 d. CAD: Chang'an decoction; ER: endoplasmic reticulum; NLRP3: nucleotide oligomerization domain-like receptor thermal protein domain associated protein 3; GRP78: glucose-regulated protein 78; PERK: protein kinase-related like endoplasmic reticulum kinase; eIF2α: eukaryotic translation initiation factor 2α; IHC: immunohistochemistry; ASC: apoptosis-associated speck-like protein containing a caspase-1 recruitment domain. Results are presented as mean ± standard deviation. aP < 0.01, dP < 0.001, fP < 0.05 vs CON; bP < 0.05, cP < 0.01, eP < 0.001 vs DSS, n = 3.

Figure 3 CAD enhanced MFN2 expression to restore ER stress and mitochondrial function A: IF staining of ROS in colon tissues; Scale bars: 20 μm; A1: CON; A2: DSS; A3: CAD-L; A4: CAD-H. B: representative electron microscopy images of colon tissues; mitochondria (Mito: pink areas) and endoplasmic reticulum (ER: yellow areas) are shown, red asterisk shows mitochondria in contact with ER; Scale bars: 1 μm; B1: CON; B2: DSS; B3: CAD-L; B4: CAD-H; C: Western blot analyses of MFN2 in colon tissues; D: statistical graphs of MFN2 relative expression level; E: IHC statistical graphs; F: IHC staining of MFN2 in colon tissues; Scale bars: 50 μm; F1: CON; F2: DSS; F3: CAD-L; F4: CAD-H. CON: control group; DSS: ulcerative coloits group; CAD-L: low-dose Chang'an decoction (0.5 g/mL); CAD-H: high-dose Chang'an decoction (2 g/mL). The drug was administered by gavage once daily for 6 d. CAD: Chang'an decoction; MFN2: mitofusin 2; ER: endoplasmic reticulum; IF: immunofluorescence; ROS: reactive oxygen species; DSS: dextran sulfate sodium; IHC: immunohistochemistry. Results are presented as mean ± standard deviation. aP < 0.001 vs CON; bP < 0.05, cP < 0.01 vs DSS, n = 3.

Figure 4 MFN2 mediated ER stress and mitochondrial damage A: representative electron microscopy images of Caco-2 cells; mitochondria (Mito: pink areas) and endoplasmic reticulum (ER: yellow areas) are shown, red asterisk shows mitochondria in contact with ER; Scale bars: 1 μm; A1: shCtrl; A2: shMFN2; A3: MFN2; shCtrl: plasmid vector group; shMFN2: MFN2 knockdown group; MFN2: MFN2 overexpression group. Cells were respective incubated with plasmid vectors for 48 h. B: Western blot analyses of GRP78, NLRP3 in Caco-2 cells; C: statistical graphs of GRP78, NLRP3 relative protein levels; D: relative mRNA expressions of CHOP, GRP78 and NLRP3 in Caco-2 cells; E: Western blot analyses of GRP78, NLRP3 in Caco-2 cells; F: statistical graphs of GRP78, NLRP3 relative protein levels; G: relative mRNA expressions of CHOP, GRP78 and NLRP3 in Caco-2 cells. Results are presented as mean ± standard deviation. aP < 0.05, bP < 0.01 vs shCtrl/Vector, n = 3. MFN2: mitofusin 2; ER: endoplasmic reticulum; GRP78: glucose-regulated protein 78; NLRP3: nucleotide oligomerization domain -like receptor thermal protein domain associated protein 3; CHOP: CCAAT enhance-binding protein homologous protein.

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