key: cord-0698775-yxnd9ebm
authors: Niu, Yaoyun; Chen, Yang; Sun, Pengbo; Wang, Yangyang; Luo, Jingyi; Ding, Yipei; Xie, Weidong
title: Intragastric and atomized administration of canagliflozin inhibit inflammatory cytokine storm in lipopolysaccharide-treated sepsis in mice: a potential COVID-19 treatment
date: 2021-05-09
journal: Int Immunopharmacol
DOI: 10.1016/j.intimp.2021.107773
sha: 40357732adf11d95e34b6626c0b7db43946ebdf9
doc_id: 698775
cord_uid: yxnd9ebm

To date, drugs to attenuate cytokine storm in severe cases of Corona Virus Disease 2019 (COVID-19) are not available. In this study, we investigated the effects of intragastric and atomized administration of canagliflozin (CAN) on cytokine storm in lung tissues of lipopolysaccharides (LPS)-induced mice. Results showed that intragastric administration of CAN significantly and widely inhibited the production of inflammatory cytokines in lung tissues of LPS-induced sepsis mice. Simultaneously, intragastric administration of CAN significantly improved inflammatory pathological changes of lung tissues. Atomized administration of CAN also exhibited similar effects in LPS-induced sepsis mice. Furthermore, CAN significantly inhibited hypoxia inducible factor 1α (HIF-1α) and phosphofructokinase-2/fructose-2,6-bisphosphatase 3 (PFKFB3) protein levels in LPS-treated lung tissues. These results indicated that CAN might attenuate cytokine storm and reduce the inflammatory symptoms in critical cases in COVID-19. Its action mechanism might involve the regulation of HIF-1α and glycolysis in vivo. However, further studies about clinical application and mechanism analysis should be validated in the future.

COVID-19 patients with pneumonia caused by new coronavirus known as SARS-Cov-2 by WHO was first found in Wuhan, China, which leads to a pandemic primarily because of rapid human-to-human transmission by respiratory tract infections and high mortality after lung infection [1, 2] . The initial mortality rate of COVID-19 declared by WHO was 2% [3] . In particular, COVID-19 has higher severity and has higher mortality in elderly patients when they have comorbidities with diabetes and cardiovascular diseases [3] [4] [5] [6] [7] .

COVID-19 pathogenesis could occur in three sequential phases: pulmonary, proinflammatory, and prothrombic [8] . Severe inflammatory lung injury is an important cause for critical cases of COVID-19 [9] . COVID-19 in critically ill patients accompanies an unprecedented spike in cytokines levels known as cytokine release syndrome and results in inflammation, infiltration of macrophages, neutrophils and lung injury in patients [10] .

COVID-19 patients with basic diseases such as diabetes and cardiovascular diseases are easily subjected to this lung injury [10] . Anti-inflammatory strategies have emerged to serve as an important method to combat the cytokine storm and improve clinical outcomes in severe COVID-19 patients. However, ideal drugs are still not available to date. Canagliflozin (CAN) is a low-molecular-weight hypoglycemic compound. It can inhibit the reabsorption of glucose by inhibiting sodium glucose cotransporter 2 (SGLT2) in renal proximal convoluted tubules; thus, glucose can be excreted in urine, and blood glucose can be reduced [11] . New studies have found that CAN has been clinically found to significantly reduce the incidence of diabetic cardiovascular events such as heart failure [12] . Considerable studies have found that CAN has new SGLT2-independent activities [13, 14] . In our previous study, we found that CAN significantly inhibited the production of inflammatory interleukin 6 (IL-6), interleukin 1 (IL-1), and tumor necrosis factor α (TNF-α) and the pathological changes of lung inflammation induced by LPS in mice [15] .

However, we did not systematically evaluate the effects of CAN on inflammatory cytokine storm.

Considering that unavailability of specific drugs to combat cytokine storm in COVID-19 patients, in this study, we would systematically investigate whether intragastric and atomized administration of CAN inhibited inflammatory cytokine storm in lung tissues of LPS-induced mouse model, which was frequently found in COVID-19 patients. The other administration was performed by a simple atomized drug delivery device.

The mice were divided into two groups. We dissolved 4 mg/mL of CAN in 50% (v/v) ethanol. The mice were placed in a sealed 1.5 L container and CAN was sprayed for 20 min at a dose of 0.4 mL/mouse. Normal control mice were given 50% ethanol at identical volume.

All mice were treated with drugs or vehicles for 3 days. After treatment, the mice were intraperitoneally injected with LPS at a dosage of 10 mg/kg for systematic inflammatory stimulation. After 4 h of LPS induction, anesthesia was induced with isoflurane and blood was collected. Immediately, the mice were sacrificed by cervical dislocation. The lung tissues were removed and weighed. Part of lung tissue aliquots were stored at −80 °C for further biochemical analysis after instantaneous freezing by liquid nitrogen. The remaining lung tissue aliquots were soaked in formalin solution and sliced for hematoxylin−eosin (H & E) staining, immunohistochemistry staining against CD11b antibody (Servicebio, Wuhan, China) and pathological analysis.

After 3 days of intragastric administration of drugs, LPS (10 mg/kg) was injected intraperitoneally in mice. After 4 h of LPS stimulation, Evans blue (1%, g/mL, Sigma Aldrich, USA) was intravenously injected at a dose of 1 mL/10 g body weight). After 10 min of Evans blue injection, lung tissue perfusion was conducted with PBS and whole lung tissues were weighed and immersed in 2 mL of formamide solution. After 24 h, the Evans blue absorbance values were detected at 620 nm. The Evans blue absorbance value and lung weight ratio were compared among different groups. 

Lung tissues (approximately 100 mg) from mice were homogenized in 1ml of ice- 

All data were presented as mean ± SD. Unpaired t-test (two tailed) was used when comparing two groups whereas one-way ANOVA with Tukey's post hoc test was used for multiple comparisons by using GraphPad Prism v8 software. P<0.05 indicated a statistically significant difference.

LPS can activate systematic inflammation and pulmonary edema. In this study, after 4 h of LPS injection (10 mg/mL), lung weight was significantly increased by 14.2% in LPS-induced mice compared with that in normal controls ( Figure 1A ). However, CAN significantly inhibited the increase by −10.1% in lung tissues, whereas DXM inhibited the increase by −18.0%. The effects of CAN were comparable to that of DXM.

Systematic inflammation may increase lung capillary permeability. Evans blue was used to evaluate this permeability after intravenous injection. We found that LPS significantly increased the Evans blue level by 95.6% in lung tissues compared with the normal control ( Figure 1B) . However, after intragastric administration, CAN significantly inhibited the increase of Evans blue concentration by −32.4% in lung tissues compared with the untreated LPS control, whereas DXM inhibited the increase by −38.5%. The effects of CAN were comparable to that of DXM.

Similarly, after atomized administration, CAN also significantly inhibited the lung weight index by −31.5% and Evans blue concentration by −31.6% in lung tissues.

Collectively, these results indicate that CAN could attenuate inflammatory lung injury. 

We assayed the pathological changes in lung tissues. The results showed that LPS significantly increased pathological changes (inflammatory cell or blood cell infiltration, alveolar septum thickening) but intragastric administration of CAN significantly inhibited the pathological changes (Figures 2A−D) . The effects of CAN were comparable to those of DXM. After atomized administration, CAN also significantly inhibited pathological changes ( Figures 2E−F) . These results indicated that CAN may reduce inflammatory injury induced by LPS in lung tissues. Figure 5A2−R2) . These results indicated that CAN might be used to attenuate inflammatory cytokine storm, inhibit inflammatory cytokine release, and reduce inflammatory lung injury through local airway application.

However, more studies should be validated. 

Glycolysis plays an important role in regulating immune cell activation and inflammatory cytokine release [18] . HIF-1α is a key factor to control glycolysis and inflammatory cytokine storm [19] . In this study, we determined the protein levels of HIF-1α and its downstream protein PFKFB3 through Western blotting. We found that LPS significantly increased HIF-1α and PFKFB3 protein levels by 203.4% and 133.4%, respectively ( Figure   6 ). However, CAN significantly attenuated the increase of HIF-1α and PFKFB3 protein levels by −53.4% and −36.6%, respectively. DXM also significantly attenuated the increase of HIF-1α and PFKFB3 protein levels by −44.1% and −37.3%, respectively. 

In COVID-19 patients, bilateral alveoli showed diffuse changes with fibrous mucoid exudates and both lungs showed interstitial lung inflammatory infiltrates with mononuclear cells and lymphocyte [20] . Intensive care unit (ICU) cases showed a significant increase of IL-6, IL-2, IL-7, IL-10, GSCF, IP-10, MCP1, MIP-1A and TNF-α levels compared with non-ICU controls [21] , which be known as "cytokines storm".

Cytokine storm indicates that the production of inflammatory cytokines such as IL-1, IL-6, and TNF-α is not controlled by immune cells [22] . Such sytokines can cause injury to all cells, causing extensive injury to the body; thus, patients will have multiple organ failure and severe diseases. COVID-19 patients may show a cytokine storm and inflammatory injuries in multiple organs, for example, the lungs, hearts, livers, and kidneys.

Cytokine storm plays an important role in the switching of disease from slight to severe [23] . Early inhibition of the production of inflammatory cytokines is an important strategy for the prevention and treatment of severe diseases. Clinical immunologists try to use glucocorticoids, IL-6 inhibitors, IL-1 inhibitors, TNF inhibitors, JAK inhibitors, chloroquine and hydroxychloroquine to suppress inflammation caused by SARS-COV-2 [24] .

However, at present, cytokine storm has no ideal drug [22] . Glucocorticoid has a significant anti-inflammatory effect, which can save the lives of many patients with COVID-19 [25] . It also plays an important role during SARS in 2003, but the related side effects are also worthy of attention [26] . The application of glucocorticoid may delay the clearance of SARS-COV-2, and lead to asymptomatic infection and secondary spread of the disease [27] . Furthermore, glucocorticoid can increase blood glucose, blood pressure, and osteoporosis. It is not suitable for some elderly patients with basic diseases such as diabetes, hypertension and osteoporosis. It may easily induce drug-induced diseases and death. A non-hormonal anti-inflammatory drug is required for the treatment of COVID-19 patients.

Recent studies have found that IL-6 is the key inflammatory factor in COVID-19 patients with inflammatory storm [28] . Therefore, blocking the IL-6 pathway is an important treatment strategy to inhibit cytokine storm [29] . Torzumab, produced by Roche, Switzerland, is an antibody against IL6 receptor, which can inhibit the inflammatory signal pathway caused by IL-6 [30] . In addition, inflammatory cytokine storm involves the release of many inflammatory factors, such as IL-1, TNF-α, and CCL-2. These factors can directly cause inflammation independent of IL6 pathway. Therefore, in theory, controlling inflammation by blocking a certain inflammatory cytokine pathway is difficult. Our research idea should anchor the master switch or more upstream link of the production control of all inflammatory factors, which may be the key to the control of systemic inflammatory storm.

Glycolysis plays a key role in the control of the production of inflammatory factors in immune cells [18] . 2-DG, a small molecule inhibiting glycolysis, significantly inhibited the production of inflammatory factors [15, 31] . In our previous study, CAN may inhibit the production of inflammatory factors by inhibiting the aerobic glycolysis metabolism of immune cells using PFKFB3 [15, 32] . Here, we systematically evaluate the effects of intragastric or atomized administration of CAN on multiple inflammatory factors and found that a significant and wide inhibition effect was found on these inflammatory factors, for example, GM-CSF, IFN-γ, IL-1α, IL-1, IL-4, IL-6, IL-7, IL-12, IL-17a, LIX, MCP-1, MIP-2 and TNF-α in lung tissues or serum. However, whether such potential mechanisms in vivo were related to the regulation of glycolysis remained unclear.

HIF-1α is a key factor involved in the regulation of glycolysis [33] . PFKFB3 is a key factor involved in glycolysis and is a downstream factor regulated by HIF-1α [34] . HIF-1α promotes glycolysis and activation of macrophages [19] . In addition, HIF-1α directly mediates the production of inflammatory factors such as IL-1 [35] . HIF-1α deficiency attenuates inflammatory damage [36] . In COVID-19, the virus causes the inflammatory sites to produce a hypoxic microenvironment and induce HIF-1α production, which may activate cytokine storm [37] . HIF-1α is used as a potential therapeutic target for acute lung injury [38] . HIF-1α/glycolysis involve SARS-CoV-2 infection and monocyte response [39] .

Therefore, HIF-1α is a potential target for the inflammatory control for COVID-19. In this study, we found that CAN significantly attenuated the increased levels of HIF-1α and its downstream PFKFB3 proteins induced by LPS. DXM also significantly inhibited the HIF-1α level as described previously [40] . These results indicated that CAN might attenuate the inflammatory cytokine storm by inhibiting the HIF-1α levels in vivo and then downregulating glycolysis in lung tissues. However, the mechanism of CAN in inhibiting HIF-1α levels should be further investigated in the future.

In this study, CAN significantly improved acute lung injures induced by LPS (increased lung edema and lung capillary penetration) in mice. The pathological slice results also indicated this improvement. Immunohistochemistry analysis further indicated that CAN attenuated macrophage (identified by CD11b antibody) infiltration in lung tissues induced by LPS, which may be associated with the decreased chemotactic cytokines, for example, MCP-1 and MIP-2 in lung tissues of mice. In blood samples, CAN significantly reduced the inflammatory factors, which suggested that CAN had a systematic inhibition on immune cell activation in vivo after acute treatment. In this study, although we used CAN or DXM only for 3 days, more COVID-19 patients may require longer administration of these anti-inflammatory drugs and a safety concern could be considered.

In our preliminary experiment, chronic administration (about 3 months) of CAN (25 mg/kg) did not affect the spleen weight in mice compared with untreated controls, whereas 1month administration of dexamethasone (2 mg/kg) significantly lowered the spleen weight in mice (supplemental figure 1) compared with untreated controls. These results indicated that CAN might not significantly affect immune organ development similar to glucocorticoids if patients required a long-term administration. However, more studies should be validated in the future. Collectively, CAN could serve as a good inhibitor of inflammatory cytokine storm in a non-cortisone manner.

This study provided a valuable reference for the treatment of severe cases of COVID-19 and inflammatory cytokine storm. At present, the drug has been on the market for the treatment of diabetes, which has a cardiovascular protective effect, with relatively few side effects. COVID-19 patients with cytokine storm and basic diseases (diabetes, hypertension and cardiovascular diseases) might benefit more from CAN treatment than other drugs. However, given the lack of ideal animal models for COVID-19, here, we induced cytokine storm in animal lungs only by LPS stimulation to simulate the clinical "cytokine storm" of COVID-19. Clinical trials should be conducted in COVID-19 patients with or without diabetes, hypertension and cardiovascular disease because CAN has direct antiinflammatory effects in vivo.

We found that intragastric administration of CAN attenuated lung tissue injury and inflammatory cytokine storm in LPS-induced mice. Atomized administration also significantly inhibited the production of inflammatory cytokines. These results indicated that CAN had a wide, non-cortisone anti-inflammatory effect, which might inhibit inflammatory cytokine storm and serve as a candidate drug to treat COVID-19 patients.

The action mechanism of CAN might be associated with the inhibition of HIF-1α/glycolysis in lung tissues. Considering that this drug could treat some basic diseases, for example, diabetes, hypertension and cardiovascular diseases, CAN might be used for t COVID patients with basic diseases; however, further validation should be conducted in the future.

This work was supported by Shenzhen Science and Technology Innovation Committee (JSGG20200519160752002) and the National Natural Science Foundation of China (No. 81373460).

Pengbo Sun, Yaoyun Niu, Yang Chen, Yangyang Wang, Jing Yi Luo, Yipei Ding contributed investigation and data analysis. Weidong Xie contributed investigation, project administration, supervision, conceptualization, data analysis, manuscript writing and revision.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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