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Journal of Central South University Medical Sciences logoLink to Journal of Central South University Medical Sciences
. 2023 Jun 28;48(6):809–820. doi: 10.11817/j.issn.1672-7347.2023.220634

Ozonated triglyceride protects against septic lethality via preventing the activation of NLRP3 inflammasome

酸化脂肪酸酯通过抑制NLRP3炎症小体活化减少脓毒症致死（英文）

WANG Dan 1,2,1, LIU Yuanhong 1,2, ZONG Xiule 1,2, YAN Siyu 1,2,✉, LU Jianyun 1,2,✉
Editor: CHEN Liwen
PMCID: PMC10930436  PMID: 37587065

Abstract

Objective

Sepsis is a critical dysregulated host response with high mortality and current treatment is difficult to achieve optimal efficacy. Ozone therapy has been revealed to protect infection and inflammation-related diseases due to its role in antibiotic and immunoregulatory effect. Ozonated triglyceride is a key component of ozonated oil that is one of ozone therapy dosage form. However, the potential role of ozonated triglyceride in sepsis remains unclear. This study aims to explore the effect of ozonated triglyceride on septic mouse model and the molecular mechanism.

Methods

Intraperitoneal injection of lipopolysaccharide (LPS), cecal ligation and puncture (CLP) were applied to construct septic mouse model. The mouse serum was obtained for detection of cytokines, and lung tissues were collected for hematoxylin and eosin (HE) staining to evaluate the extent of lung injury in septic mouse with ozonated triglyceride treatment at different time and doses. The survival of septic mice was observed for 96 h and Kaplan-Meier analysis was used to analyze the survival rates. In addition, primary peritoneal macrophages and human acute monocytic-leukemia cell line (THP-1) were treated with inflammasome activators with or without ozonated triglyceride. The level of cytokines was detected by enzyme-linked immunosorbent assay (ELISA). The cleavage of caspase-1 and gasdermin-D (GSDMD) was detected by Western blotting.

Results

Ozonated triglyceride at different time and doses reduced the release of inflammasome-related cytokines [interleukin (IL)-1β and IL-18] (all P<0.05) but not pro-inflammatory cytokines such as IL-6 and tumor necrosis factor-α (TNF-α) in septic mice (all P>0.05). Ozonated triglyceride significantly improved the survival rate of septic mice and reduced sepsis-induced lung injury (all P<0.05). Ozonated triglyceride significantly suppressed the canonical and non-canonical activation of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome (all P<0.05) but not affected absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) inflammasomes in vitro (all P>0.05). Ozonated triglyceride reduced the cleavage of caspase-1 and the downstream GSDMD.

Conclusion

Ozonated triglyceride presents a protect effect on sepsis lethality via reducing cytokines release and sepsis-related organ injury. The mechanism is that ozonated triglyceride specifically suppresses the activation of NLRP3 inflammasome. Ozonated triglyceride is a promising candidate for sepsis treatment.

Keywords: sepsis, ozonated triglyceride, NLRP3 inflammasome, ozone therapy, ozonated oil

Sepsis is defined as a dysregulated host response to infection and may eventually lead to life-threatening organ dysfunction[1]. Sepsis is a complex syndrome characterized by physiological, pathological, and biochemical abnormalities. Sepsis presents rapid progression and high mortality, which is a critical illness worldwide. A total of 48.9 million sepsis patients were identified worldwide in 2017, with a mortality rate of more than 20%[2-3]. The incidence of sepsis continues to increase significantly, and the medical costs are enormous. Statistics show that the annual cost of sepsis treatment in the United States is more than $20 billion[4]. Thus, sepsis is a serious public health problem. Currently, antibiotics, fluid resuscitation, and multiple organ support therapies were applied to sepsis patients, but the efficacy is limited which is hard to reach satisfactory remission. Therefore, novel, safe, and effective treatments are urgently needed in the clinic for sepsis patients.

The clinical application of ozone therapy has received a lot of attention since its first applied for patients during the First World War. Much of our team’s previous work focused on the role of ozone therapy in infection and inflammation-related diseases. The previous studied identified that ozone therapy could improve skin bacterial[5-6] or fungal infections[7], as well as treat inflammatory diseases including psoriasis[7] and atopic dermatitis[8]. At the same time, the safety of ozone therapy had already been evaluated[9]. Current ozone medical preparation in clinical practice mainly classified as ozone hydrotherapy, ozonated oil externally used, and ozone autohemotherapy (OAHT)[10]. Ozonated oil is a product rich in unsaturated fatty acids obtained by ozonizing basic oil (camellia oil) with ozone. Ozonated triglyceride, also named as acidified aliphatic ester, is the key component of ozonated oil, and the range of acidity value and peroxide value are 4 to 15 mg/g and 160 to 240 Meq/kg, respectively[11]. Combined with previous study[11] confirming that ozone therapy improved infection and inflammation-related diseases and sepsis is a typical disease related to both infection and inflammasome, we speculate whether ozonated triglyceride, a key component of ozone therapy, could treat sepsis or not.

In this study, to explore the effect of ozonated triglyceride in sepsis mouse model and the molecular mechanism, 2 classical types of septic mouse models, including lipopolysaccharide (LPS)-induced endotoxemia and cecal ligation and puncture (CLP)-induced peritonitis, were constructed. Cytokines release, lung-tissue injury and survival rate were detected and analyzed. Besides, primary peritoneal macrophages and human acute monocytic-leukemia cell line (THP-1) were applied to explore the potential mechanisms. This study focuses on the current dilemma of sepsis treatment and aims to find novel therapy for sepsis.

1. Materials and methods

1.1. Septic mouse model

Eight-week-old C57 male mice were applied for the construction of the septic mouse model. LPS (20 mg/kg) was intraperitoneally injected to induce endotoxemia and CLP was performed to induce sepsis. Mice were anesthetized and the abdomen was incised for CLP. Then the cecum was isolated and ligated, and the cecum was punctuated with 2 holes to facilitate its contents into the enterocoelia. For enzyme-linked immunosorbent assay (ELISA) assays, 50, 100 or 200 μL ozonated triglyceride (0.736 g/mL) was intraperitoneally injected 1 h before or after the sepsis model construction, and the blood was sampled from mouse eyes 14 h later. For the survival assays, 100 μL ozonated triglyceride was intraperitoneally injected once 1 h after the septic model construction. This study was approved by the Ethics Committee of the Third Xiangya Hospital, Central South University (XMSB-2022-0296). All procedures were conducted in accordance with the Institutional Animal Care and Use Committee of Central South University.

1.2. Hematoxylin and eosin staining

To evaluate the severity of sepsis-induced lung injury, 14 h after the sepsis model construction, lung tissues were collected and incubated in paraffin after the death of mice. The paraffin was sliced into 5-mm thick sections and stained with hematoxylin and eosin (HE) staining.

1.3. Cell culture

Primary peritoneal macrophages were extracted from 8-week-old male C57 mice. Mice were intraperitoneally injected with 3% thioglycolate, and peritoneal exudate cells were harvested and incubated after 72 h. Non-adherent cells were removed 1 to 2 h later, and the adherent monolayer cells were mainly peritoneal macrophages. Roswell Park Memorial Institute (RPMI) 1640 medium with 10% fetal bovine serum was used to cultured THP-1 cell line, and 100 nmol/L phorbol-12-myristate-13-acetate (PMA) treated for 3 h to differentiate THP-1 cell line for subsequently treatment.

1.4. Stimulation of NLRP3 inflammasome

Cells (5×104) were incubated with 5 mmol/L adenosine triphosphate (ATP) for 1 h or 10 μmol/L nigericin for 1 h following 100 ng/mL LPS for 3 h to activate NLRP3 inflammasome in canonical pathway. LPS (2 μg/mL) was combined with 5 μg/mL cholera toxin subunit B (CTB+LPS) or LPS transfection by FuGENE HD reagent (Promega, USA) for 16 h to activate NLRP3 inflammasome in non-canonical pathway. Poly(dA:dT) (1 μg/mL) was transfected into cytoplasm to activate absent in melanoma 2 (AIM2) inflammasome and 2 μg/mL flagellin (FLA) for 1 h following 100 ng/mL LPS for 3 h. Ozonated triglyceride with 0.5 or 1 μL was treated at the beginning of the induction.

1.5. Western blotting

Radio immunoprecipitation assay (RIPA) buffer (Beyotime, China) was applied to lyse cells, and the protein was separated in 10% sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE) gel. After being transferred onto the polyvinylidene fluoride (PVDF) membranes, the membrane was blocked with 5% non-fat milk for 1 h. The protein content was detected using an electrochemiluminescence (ECL) kit (Pierce, USA) after incubating with primary and secondary antibodies step by step. Primary antibodies used in this study were as follows: Caspase-1 (ab179515; Abcam, UK), NLRP3 (Cryo-2, AdipoGen Switzerland), gasdermin-D (GSDMD) (ab209845, Abcam, UK), and β-actin (8H10D10, CST, USA).

1.6. ELISA

ELISA kits (eBioscience or Abcam, UK) were applied to detect the level of interleukin (IL)-1β, IL-18, IL-6, and tumor necrosis factor-α (TNF-α) according to the manufacturer’s instructions.

1.7. Statistical analysis

Data were analyzed with GraphPad Prism 8.0 and were presented as mean±standard deviation. The differences between the 2 groups were analyzed with Student’s t-test, and the comparison of more than 2 groups used one-way ANOVA test. Survival differences between the 2 groups were analyzed by Kaplan Meier. P<0.05 was considered statistically significant.

2. Results

2.1. Ozonated triglyceride inhibits the release of cytokines in sepsis in vivo

ELISA results showed that 50, 100, and 200 µL of ozonated triglyceride significantly reduced the release of IL-1β. There was no significant difference between the dosage effects of 100 and 200 µL (Supplementary Figure 1, https://doi.org/10.11817/j.issn.1672-7347.2023. 220634F1). Therefore, we selected doses of 50 and 100 µL of ozonated triglyceride for subsequent detection of inflammatory cytokines. Ozonated triglyceride was applied to mice 1 h before or after the septic model construction with 2 different dosages, 50 or 100 µL. The results showed that ozonated triglyceride significantly reduced the release of IL-1β and IL-18 no matter ozonated triglyceride dose and treatment time, whereas the release of TNF-α and IL-6 was not affected in LPS-induced endotoxemia (Figure 1A) and CLP-induced sepsis (Figure 1B).

Figure 1. Ozonated triglyceride inhibits the release of cytokines in sepsis in vivo.

Figure 1

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Eight-week-old C57 mice were intraperitoneally injected with 20 mg/kg LPS (A) or CLP was constructed (B) to induce sepsis model, and ozonated triglyceride was treated with 1 h before or after the sepsis model construction with different doses (50 or 100 µL). The level of IL-1β, IL-18, IL-6 and TNF-α were detected by ELISA 14 h after sepsis model construction. *P<0.05, **P<0.01, ***P<0.001, n=5. LPS: Lipopolysaccharide; CLP: Cecal ligation and puncture; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α; ELISA: Enzyme-linked immunosorbent assay; VC: Vehicle control; OT: Ozonated triglyceride; OT-1: 50 µL Ozonated triglyceride; OT-2: 100 µL Ozonated triglyceride.

2.2. Ozonated triglyceride prevents septic lethality and sepsis-induced lung injury in vivo

Based on the above results, ozonated triglyceride was chosen at a dose of 100 µL, 1 h after sepsis model construction. The results showed that ozonated triglyceride treatment significantly prompted the survival rate of septic mice (Figure 2A, 2B; all P<0.05). Moreover, ozonated triglyceride significantly attenuated endotoxemia-induced lung injury in LPS-induced endotoxemia (Figure 2A, P<0.001) and CLP-induced sepsis (Figure 2B, P<0.001).

Figure 2. Ozonated triglyceride improves the survival of sepsis mice and reduces lung injury in vivo.

Figure 2

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A: Survival of LPS-induced endotoxemia mice with or without ozonated triglyceride (100 µL) 1 h after sepsis model construction was observed for 96 h. n=8. HE staining of lung tissue sections. Bars=100 μm, n=5. Fold change of lung injury score. Briefly, morphological changes were scored as nil (0), mild (1), moderate (2), or severe (3) injury based on the presence of exudates, hyperemia or congestion, infiltration of neutrophils, alveolar hemorrhage, presence of debris, and cellular hyperplasia, in a blinded fashion. The fold changes of the sums of scores were plotted on a bar graph. B: Survival of CLP-induced sepsis mice with ozonated triglyceride treatment was observed for 96 h. n=8. HE staining of lung tissue sections and fold change of lung injury score. n=5. *P<0.05, ***P<0.001. LPS: Lipopolysaccharide; CLP: Cecal ligation and puncture; HE: Hematoxylin and eosin; VC: Vehicle control; OT: Ozonated triglyceride.

2.3. Ozonated triglyceride specifically inhibits the activation of canonical NLRP3 inflammasome in vitro

The results showed that ozonated triglyceride significantly reduced the release of IL-1β and IL-18 on primary peritoneal macrophage (Figure 3A) or THP-1 cell line (Figure 3B) in a dose-dependent manner (all P<0.05), while did not change the level of TNF-α and IL-6 (all P>0.05).

Figure 3. Ozonated triglyceride reduces canonical activation of NLRP3 inflammasome.

Figure 3

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Canonical activation of NLRP3 inflammasome was stimulated using LPS combined with ATP or nigericin, and the level of IL-1β, IL-18, IL-6, and TNF-α was detected after the application of ozonated triglyceride with 0.5 or 1 µL. A: Mouse peritoneal macrophages; B: THP-1 cell line. *P<0.05; **P<0.01; ***P<0.001. NLRP3: NOD-like receptor family pyrin domain containing 3; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α; VC: Vehicle control; OT-1: 0.5 µL ozonated triglyceride; OT-2: 1 µL ozonated triglyceride; Mock: Blank control; LPS: Lipopolysaccharide; Nig: Nigericin.

The results showed that ozonated triglycerides did not affect the release of IL-1β and IL-18 associated with AIM2 and NLR family CARD domain-containing protein 4 (NLRC4) inflammasome (Figure 4, all P>0.05).

Figure 4. Ozonated triglyceride do not affect the activation of AIM2 or NLRC4 inflammasome.

Figure 4

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Poly(dA:dT) was applied to activate AIM2 inflammasome and LPS+flagellin was used to activate NLRC4 inflammasome in primary peritoneal macrophages with or without ozonated triglyceride, the level of IL-1β (A), IL-18 (B), IL-6 (C), and TNF-α (D) was detected by ELISA. AIM2: Absent in melanoma 2; NLRC4: NLR family CARD domain-containing protein 4; VC: Vehicle control; OT-1: 0.5 µL ozonated triglyceride; OT-2: 1 µL ozonated triglyceride; Mock: Blank control; LPS: Lipopolysaccharide; FLA: Flagellin; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α; ELISA: Enzyme-linked immunosorbent assay.

2.4. Ozonated triglyceride inhibits the activation of non-canonical NLRP3 inflammasome

The results showed that ozonated triglyceride suppressed IL-1β and IL-18 release in macrophages (Figure 5A) and THP-1 cell line (Figure 5B) in a dose-dependent manner (all P<0.05) without affecting TNF-α and IL-6 levels (all P>0.05).

Figure 5. Ozonated triglyceride reduces non-canonical activation of NLRP3 inflammasome.

Figure 5

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Non-canonical activation of NLRP3 inflammasome was conducted by transfecting LPS using FuGENE reagent or CTB, and the level of IL-1β, IL-18, IL-6, and TNF-α was detected after the application of ozonated triglyceride with 0.5 or 1 µL. A: Mouse peritoneal macrophages; B: THP-1 cell line. **P<0.01; ***P<0.001; NLRP3: NOD-like receptor family pyrin domain containing 3; VC: Vehicle control; OT-1: 0.5 µL ozonated triglyceride; OT-2: 1 µL ozonated triglyceride; Mock: Blank control; CTB: Cholera toxin subunit B; LPS: Lipopolysaccharide; IL-1β: Interleukin-1β; TNF-α: Tumor necrosis factor-α.

2.5. Ozonated triglyceride inhibits the cleavage of caspase-1 and GSDMD

Western blotting results showed that ozonated triglyceride reduced caspase-1 and IL-1β cleavage upon canonical NLRP3 inflammasome stimulus (Figure 6A) and suppressed GSDMD cleavage (Figure 6B). The administration of ozonated triglyceride was found to decrease caspase-1 and GSDMD cleavage in the lung tissues of the LPS-induced sepsis mouse model (Figure 6C).

Figure 6. Ozonated triglyceride reduces the cleavage of caspase-1 and the downstream GSDMD.

Figure 6

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A: Cleavage of caspase-1 and IL-1β was detected by Western blotting under canonical NLRP3 inflammasome stimulus with or without ozonated triglyceride. B: Cleavage of GSDMD was detected under canonical NLRP3 inflammasome stimulus with or without ozonated triglyceride. C: Cleavage of caspase-1 and GSDMD was reduced under ozonated triglyceride treatment in lung tissues of LPS-induced sepsis model. Similar results were obtained from 3 independent experiments. Casp1: Caspasc-1; NLRP3: NOD-like receptor family pyrin domain containing 3; GSDMD: Gasdermin-D; IL-1β: Interleukin-1β; VC: Vehicle control; OT: 1 µL ozonated triglyceride; Mock: Blank control; LPS: Lipopolysaccharide; Nig: Nigericin; ATP: Adenosine triphosphate; FL: Full-length; N: N domain.

3. Discussion

Our study revealed that ozonated triglyceride reduced the sepsis-related cytokines, improved the survival of septic mice, and protected sepsis-induced lung injury in vivo. Further investigation showed ozonated triglyceride inhibited the activation of canonical and non-canonical NLRP3 inflammasome, while had little effect on AIM2 and NLRC4 inflammasome. In addition, ozonated triglyceride inhibited caspase-1 and downstream GSDMD cleavage upon canonical NLRP3 inflammasome stimulus. Therefore, the results showed that ozonated triglyceride could ameliorate septic lethality via inhibiting NLRP3 inflammasome activation, which suggests an alternative strategy for sepsis therapy.

Currently, ozone therapy is widely used in the prevention and treatment of multiple diseases. Ozone therapy has been shown to play an important role in infection-related illnesses. Ozone hydrotherapy and ozonated oil had been widely used to treat skin infections, including bacterial, viral, and fungal infections, and has proven to be significantly effective[11]. In addition, the role of ozone therapy in COVID-19 treatment attracted attention due to the study[12] suggested a possible effect of ozone therapy in SARS-CoV-2, either as a monotherapy, or as an adjunct to standard treatment regimens. SARS-CoV-2 is a new emerged virus for the past few years, so there is little exactly therapy against it. By summarizing 280 previous articles on ozone treatment for COVID-19, a narrow review[13] showed ozone exerts antiviral activity through inhibiting viral replication and direct inactivating with viruses. Study[14] showed that ozone therapy via appropriate routes and at small doses can protect severe COVID-19 induced lung injury, especially treated in early stages of the disease. Compared to standard of care (SOC) alone, ozone therapy combined with SOC improved the clinical status and reduced the viral load, led to earlier recovery for mild to moderate COVID-19 patients[15]. Besides, ozone therapy possible also play a positive role in sepsis. In septic model, ozone therapy significantly decreased inflammation and lung injury via antioxidant defenses, and supported the therapeutic effect of antibiotics[16-18]. Ozone therapy was proved to modulate the inflammatory response and protect acute lung injury in the CLP infection model in rats[19]. These studies suggest that ozone therapy plays an important role in infection-related diseases, especially in severe infection-induced lung injury. Ozonated oil is an important dosage form of ozone therapy. As a key component of ozonated oil, it is not clear whether ozonated triglyceride had an effect on sepsis. In this work, 2 classical sepsis models, LPS intraperitoneal injection and CLP were constructed, and ozonated triglyceride with different doses and time of administration. The results showed that ozonated triglyceride reduced the release of cytokines and improved survival rate of septic mice, which suggested a protective role of ozonated triglyceride in sepsis.

Next, we looked for clues from the possible mechanism of ozone therapy and the key mechanism of sepsis pathogenesis to explore the mechanism of ozonated triglyceride protection of sepsis. Except for antibacterial and antioxidant effects, ozone therapy is proven to have an immunoregulatory effect. Ozone therapy induced the release of various cytokines to activate immune functions[10]. The over-activated NLRP3 inflammasome had been demonstrated to play a key role in the pathogenesis of sepsis. Inflammasomes are complex groups of multimeric proteins that induced the release of IL-1β and IL-18 in response to stimuli, and NLRP3 inflammasome is the most studied one. The expression of NLRP3 inflammasome was upregulated in macrophages of sepsis patients[20]. Blocking the activation of NLRP3 inflammasome improved the survival rate of septic mice and attenuated sepsis-induced lung injury[21-22]. Our previous work[23] found that inhibiting NLRP3 inflammasome reduced LPS-induced systemic inflammation and lung injury. Another previous study[24] showed CEFFE, an extractive from nanofat, protected septic mobility and organ injury in mice via targeting NLRP3 inflammasome. Meanwhile, our results showed ozonated triglyceride selectively decreased the inflammasome-related cytokines in septic mice. Therefore, the mechanism of ozonated triglyceride protected sepsis might be related to NLRP3 inflammasome. NLRP3 inflammasome could be activated via canonical pathway and non-canonical pathway stimulated by LPS in cytoplasm[25-26]. The results subsequently in vitro showed ozonated triglyceride reduced the release of inflammasome-related cytokines under canonical or non-canonical NLRP3 inflammasome stimulus in primary peritoneal macrophage and THP-1 cell line in a dose-dependent manner. The AIM2 and NLRC4 inflammasomes share a similar structure with the NLRP3 inflammasome and are also capable of inducing an elevation of IL-1β and IL-18. In order to investigate the effect of ozonated triglyceride on these inflammasomes, we activated the AIM2 and NLRC4 inflammasomes using poly (dA:dT) and flagellin respectively. Compared to NLRP3 inflammasome, the application of ozonated triglyceride failed to reduce the release of IL-1β and IL-18 after the activation of AIM2 and NLRC4 inflammasomes. The results suggested that ozonated triglyceride protected sepsis via targeting NLRP3 inflammasome.

The activated NLRP3 inflammasome on the one hand could cleave caspase-1 and subsequently induce the release of IL-1β and IL-18; on the other hand, it could cleave the downstream GSDMD to GSDMD-N, and induce cell swelling and rupture, lead to pyroptosis[26-27]. Therefore, whether ozonated triglyceride affect the cleavage of caspase-1 and downstream GSDMD should be identified. The results showed that ozonated triglyceride decreased the cleavage of caspase-1 and IL-1β as well as GSDMD, which suggested that ozonated triglyceride impacted on the downstream signal of the activation of NLRP3 inflammasome.

In conclusion, the work revealed that ozonated triglyceride, the key ingredient of ozonated oil, could significantly ameliorate sepsis mobility and sepsis-induced injuries by suppressing NLRP3 inflammasome activation. Therefore, ozonated triglyceride is a potential ideal approach for sepsis therapy.

Acknowledgments

We would like to thank Wiley editing services for kindly reviewing and revising on English writing.

Appendix.

Supplementary Figure 1 Different dose of ozonated triglyceride reduced the release of IL-1β in LPS-induced sepsis model .

Supplementary Figure 1 Different dose of ozonated triglyceride reduced the release of IL-1β in LPS-induced sepsis model

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8-Week-old C57 mice were intraperitoneally injected with 20 mg/kg LPS to induce sepsis model, and ozonated triglyceride was treated with 1h before the sepsis model construction with different doses (50, 100 or 200 µL). Fourteen hours after sepsis model construction, the level of IL-1β was detected by ELISA. **P<0.01, ***P<0.001, n=5 biologically independent mice. LPS, Lipopolysaccharide; IL-1β, Interleukin-1β; ELISA, Enzyme-linked immunosorbent assay; VC, Vehicle Control; OT, Ozonated triglyceride; OT-1, 50 µL Ozonated triglyceride; OT-2, 100 µL Ozonated triglyceride; OT-3, 200 µL Ozonated triglyceride.

Funding Statement

This work was supported by the National Natural Science Foundation (82202391), Outstanding Youth Project of Natural Science Foundation of Hunan Province (2022JJ20093), and the Wisdom Accumulation and Talent Cultivation Project of the Third Xiangya Hospital of Central South University (YX202201), China.

Conflict of Interest

The authors declare that they have no conflicts of interest to disclose.

AUTHORS’CONTRIBUTIONS

WANG Dan Research design, experimental operation, and paper writing; LIU Yuanhong, ZONG Xiule Data collecting and analysis; YAN Siyu, LU Jianyun Research design, paper supervision and revision. The final version of the manuscript has been approved and read by all authors.

Note

http://xbyxb.csu.edu.cn/xbwk/fileup/PDF/202306809.pdf

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