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. 2023 Jan 13:9:1075465.
doi: 10.3389/fmed.2022.1075465. eCollection 2022.

Cryptotanshinone attenuates LPS-induced acute lung injury by regulating metabolic reprogramming of macrophage

Zesen Ye  1 , Panxia Wang  2 , Guodong Feng  1 , Quan Wang  1 , Cui Liu  1 , Jing Lu  1 , Jianwen Chen  1 , Peiqing Liu  1
Affiliations

Cryptotanshinone attenuates LPS-induced acute lung injury by regulating metabolic reprogramming of macrophage

Zesen Ye et al. Front Med (Lausanne). .

Abstract

Background: Acute lung injury (ALI) is a life-threatening inflammatory disease without effective therapeutic regimen. Macrophage polarization plays a key role in the initiation and resolution of pulmonary inflammation. Therefore, modulating macrophage phenotype is a potentially effective way for acute lung injury. Cryptotanshinone (CTS) is a lipophilic bioactive compound extracted from the root of Salvia miltiorrhiza with a variety of pharmacological effects, especially the anti-inflammatory role. In this study, we investigated the therapeutic and immunomodulatory effects of CTS on ALI.

Materials and methods: The rat model of ALI was established by intratracheal instillation of LPS (5 mg/kg) to evaluate the lung protective effect of CTS in vivo and to explore the regulation of CTS on the phenotype of lung macrophage polarization. LPS (1 μg/mL) was used to stimulate RAW264.7 macrophages in vitro to further explore the effect of CTS on the polarization and metabolic reprogramming of RAW264.7 macrophages and to clarify the potential mechanism of CTS anti-ALI.

Results: CTS significantly improved lung function, reduced pulmonary edema, effectively inhibited pulmonary inflammatory infiltration, and alleviated ALI. Both in vivo and in vitro results revealed that CTS inhibited the differentiation of macrophage into the M1 phenotype and promoted polarization into M2 phenotype during ALI. Further in vitro studies indicated that CTS significantly suppressed LPS-induced metabolic transition from aerobic oxidation to glycolysis in macrophages. Mechanistically, CTS blocked LPS-induced metabolic transformation of macrophages by activating AMPK.

Conclusion: These findings demonstrated that CTS regulates macrophage metabolism by activating AMPK, and then induced M1-type macrophages to transform into M2-type macrophages, thereby alleviating the inflammatory response of ALI, suggesting that CTS might be a potential anti-ALI agent.

Keywords: AMPK; Cryptotanshinone; acute lung injury; macrophage polarization; metabolic reprogramming.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Cryptotanshinone (CTS) ameliorates LPS-induced acute lung injury in rats. LPS was used to induce in vivo ALI and CTS at different concentrations was administrated to rat. Representative parameters of mice pulmonary function: (A) enhanced pause (Penh), (B) end-inspiratory pause (EIP), (C) relaxation time (RT), (D) end-expiratory pause (EEP), (E) minute ventilation volume (MV), n = 6. (F) Representative HE staining results of lung histopathological changes, scale bar: 100 μm, n = 6. (G) Lung histopathological score, n = 6. (H) Lung coefficient (%) changes in each group, n = 8. (I) Wet-dry weight ratio of right lung (Lung W/D ratio) in rats, n = 6. (J) Total protein concentration in BALF, n = 5. (K) The MPO activity level in the lung tissues was measured, n = 6. ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the Model group.
FIGURE 2
FIGURE 2
Cryptotanshinone (CTS) inhibited M1-type polarization and induces M2-type polarization of macrophages in vivo. (A) The levels of IL-1β, IL-6, TNF-α, and IL-10 in BALF were determined using ELISA, n = 6. (B,C) Representative immunofluorescence image of lung tissues. DAPI (blue), CD68 (red), CD86 (green), and CD206 (green), scales: 25 μm, n = 4. (D) The protein levels of iNOS and Arg-1, n = 4. (E) Lung sections were immunohistochemically stained by anti-iNOS antibody and anti-Arg-1 antibody, n = 4. Data were presented as the mean ± SEM. ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the Model group.
FIGURE 3
FIGURE 3
Cryptotanshinone (CTS) inhibited M1-type polarization and promoted M2-type polarization of RAW264.7 macrophages. (A) CTS alone or co-treatment with or without LPS stimulation and cell viability was measured. RAW264.7 were pretreated with CTS (2.5, 5, and 10 μM) for 2 h and then co-stimulated with LPS (1 μg/mL) for another 24 h. (B) The protein expression of iNOS and CD86, n = 3. (C) The protein expression of Arg-1 and CD206, n = 3. (D) The proportion of CD86 positive cells was analyzed by flow cytometry, n = 3. (E) The proportion of CD206 positive cells was analyzed by flow cytometry, n = 3. Data were presented as the mean ± SEM. ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the LPS group.
FIGURE 4
FIGURE 4
Cryptotanshinone (CTS) blocks LPS-induced metabolic reprogramming of RAW264.7 macrophages. RAW264.7 were pretreated with CTS (2.5, 5, and 10 μM) for 2 h and then co-treated with LPS for another 24 h. (A) Glucose uptake was detected using assay kit, n = 4. (B) The level of lactic acid, n = 4. (C,D) The extracellular acidification rate (ECAR) of macrophages were measured, n = 4. (E,F) The oxygen consumption rate (OCR) was measured, n = 4. (G) The protein levels of PKM2, PFKFB3 and GLUT1 were analyzed by using western blotting assay, n = 3. Data were presented as mean ± SEM. ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the LPS group.
FIGURE 5
FIGURE 5
Cryptotanshinone (CTS) regulates RAW264.7 macrophage polarization via AMPK. (A) The protein levels of p-AMPK/AMPK in LPS-induced acute lung injury were analyzed by western blot, n = 4. (B) The protein expression of p-AMPK/AMPK in LPS-treated macrophage was analyzed by western blot, n = 3. (C-E) RAW264.7 pretreated with 5?μM AMPK inhibitor compound C for 2 h were treated with 10 μM CTS for 2?h and then exposed to LPS for another 24 h. (C) The protein expression levels of p-AMPK/AMPK were assessed by western blot analysis, n = 3. (D) The protein expression of iNOS, Arg-1, CD86, and CD206 was detected by Western blot, n = 3. (E) The proportion of CD86+ cells and CD206+ cells were analyzed by flow cytometry, n = 3. Data were presented as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the LPS group; &P < 0.05, &&P < 0.01 vs. the LPS + CTS (10 μM) group.
FIGURE 6
FIGURE 6
Cryptotanshinone (CTS) regulates energy metabolism of LPS-induced RAW264.7 macrophages through AMPK. RAW264.7 pretreated with 5 μM AMPK inhibitor compound C for 2 h were treated with 10 μM CTS for 2 h and then exposed to LPS for another 24 h. (A) ECAR of the indicated macrophages were measured with a seahorse analyzer. (B) Glycolysis, glycolytic capacity, and glycolytic reserve were calculated and are indicated as ECAR in mpH/min, n = 4. (C) OCR was measured with the Seahorse analyzer. (D) The basal respiration, maximal respiration, and ATP production were calculated and are indicated as OCR in pmoles/min, n = 4. (E) The protein level of HIF-1α was analyzed by using western blotting assay, n = 3. (F) The mRNA expressions of glucose transporter type1 (GLUT1), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), pyruvate kinase M2 (PKM2) were detected by using the qPCR assay, n = 3. (G) The mRNA expressions of carnitine palmitoyltransferase 1A (CPT1A), carnitine palmitoyltransferase 2 (CPT2), medium-chain acyl-coA dehydrogenase (MCAD) were detected by using the qPCR assay, n = 3. Data were presented as the mean ± SEM. ##P < 0.01 vs. the control group; *P < 0.05, **P < 0.01 vs. the LPS group; &P < 0.05, &&P < 0.01 vs. the LPS + CTS (10 μM) group.
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Grants and funding

All research were supported by grants from the National Natural Science Foundation of China (U21A20419 and 82104157), Natural Science Foundation of Guangdong Province (2022A1515012322), the Fundamental Research Funds for the Central Universities, Sun Yat-sen University (22qntd4510), Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program (2017BT01Y093), National Engineering and Technology Research Center for New drug Druggability Evaluation (Seed Program of Guangdong Province, 2017B090903004), Guangdong Provincial Key Laboratory of Construction Foundation (2017B030314030), and Guangdong Provincial Key Laboratory of Construction Foundation, No. 2019B030301005, Guangzhou Basic and Applied Basic Research Project (202102020173 and 202206080007), and the Discipline Construction Project of Guangdong Medical University (4SG21233G).