Baicalein self‐microemulsion based on drug–phospholipid complex for the alleviation of cytokine storm

Abstract Cytokine storm is a phenomenon whereby the overreaction of the human immune system leads to the release of inflammatory cytokines, which can lead to multiple organ dysfunction syndrome. At present, the existing drugs for the treatment of cytokine storm have limited efficacy and severe adverse effects. Here, we report a lymphatic targeting self‐microemulsifying drug delivery system containing baicalein to effectively inhibit cytokine storm. Baicalein self‐microemulsion with phospholipid complex as an intermediate carrier (BAPC‐SME) prepared in this study could be spontaneously emulsified to form 12‐nm oil‐in‐water nanoemulsion after administration. And then BAPC‐SME underwent uptake by enterocyte through endocytosis mediated by lipid valve and clathrin, and had obvious characteristics of mesenteric lymph node targeting distribution. Oral administration of BAPC‐SME could significantly inhibit the increase in plasma levels of 14 cytokines: TNF‐α, IL‐6, IFN‐γ, MCP‐1, IL‐17A, IL‐27, IL‐1α, GM‐CSF, MIG, IFN‐β, IL‐12, MIP‐3α, IL‐23, and RANTES in mice experiencing systemic cytokine storm. BAPC‐SME could also significantly improve the pathological injury and inflammatory cell infiltration of lung tissue in mice experiencing local cytokine storm. This study does not only provide a new lymphatic targeted drug delivery strategy for the treatment of cytokine storm but also has great practical significance for the clinical development of baicalein self‐microemulsion therapies for cytokine storm.


| INTRODUCTION
The cytokine storm refers to an excessive immune and inflammatory response to external stimuli, characterized by excessive proliferation of T cells and macrophages and the rapid release of inflammatory cytokin. It can be caused by many infectious and non-infectious diseases, such as influenza, coronavirus disease (COVID- 19), septicemia, and lupus erythematosus, as well as chimeric antigen receptor T cell therapy. [1][2][3][4][5] The occurrence of cytokine storm can accelerate disease progression, leading to tissue and organ damage, as well as multiple organ dysfunction syndrome. 6 Some studies have shown that reducing the occurrence of cytokine storm among patients with infectious or non-infectious diseases can help to reduce organ damage and slow down disease progression, which is particularly important in the treatment of critically ill patients ( Figure 1). 7,8 When cytokine storm result from infection, the levels of proinflammatory cytokines represented by interferon (IFN)-λ, tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-2, IL-5, IL-6, IL-7, IL-8, granulocyte colony-stimulating factor (G-CSF), granulocyte macrophage colonystimulating factor (GM-CSF), vascular endothelial growth factor (VEGF), monocyte chemoattractant protein-1 (MCP-1), IFN-γ-inducible protein-10 (IP-10), and macrophage inflammatory protein-1 (MIP-1) were significantly increased in vivo. This is also the main cause of multiple organ dysfunction and failure caused by cytokine storm. [8][9][10][11][12][13] The lymph nodes and spleen are mainly composed of T and B lymphocytes, contain a large number of macrophages and dendritic cells, and constitute the main sites of occurrence of immune reactions in vivo. [14][15][16][17][18] When the presence of certain infection, immune cells in the lymph nodes and spleen are overactivated, proliferate, differentiate, and secrete a large number of proinflammatory cytokines, thereby leading to systemic cytokine storm.
F I G U R E 1 Schematic diagram of the formation of cytokine storm and the effect of BAPC-SME. When lungs are infected by certain viruses or bacteria, macrophages and dendritic cells in the lung tissue could be over-activated. Lymphocytes, neutrophils, and other inflammatory cells would be recruited from the blood into the lung. Further, these immune cells secrete a large number of inflammatory cytokines, thus forming local cytokine storm. At the same time, these cytokines and viruses could leak into the systemic circulation and enter the mesenteric lymph nodes (the largest lymph nodes in the body) with the blood. They activate the immune cells in the lymph nodes and promote their proliferation and differentiation, resulting in the secretion of a large number of pro-inflammatory cytokines. These differentiated immune cells and proinflammatory cytokines are transported into the systemic circulation through the lymphatic system, thus causing systemic cytokine storm. This would cause inflammatory damage to tissues and organs. After oral administration, BAPC-SME was spontaneously emulsified in the gastrointestinal tract to form nanoscale emulsion droplets, which then targeted the mesenteric lymph nodes through lymphatic transport. Therefore, baicalein could directly act on the immune cells in the lymph nodes and inhibit the excessive expression of inflammatory cytokines. In addition, upon entering the systemic circulation, baicalein and its metabolite baicalin could also inhibit the secretion of cytokines by immune cells in lung tissue, and exhibited an inhibitory effect on viruses in tissues.
At present, glucocorticoids, cyclooxygenase inhibitors, TNF inhibitors, IL-1 antagonists, and IL-6 antagonists are mainly used in the treatment of cytokine storm. 4,7 However, their curative effects are limited and their adverse effects are severe. For example, glucocorticoids can nonspecifically inhibit cytokine storm; however, their longterm use can easily cause adverse effects such as double infections, diabetes, osteoporosis, hypertension, and osteonecrosis. Therefore, the development of new safe and effective drugs that regulate cytokine storm is of great significance to save patients' lives.
Scutellaria baicalensis has been used as a traditional Chinese medicinal plant for thousands of years and has a significant immunocgqzressive effect. 19 Baicalein and baicalin are the main bioactive components extracted from the root of S. baicalensis. [20][21][22] At present, a large number of studies have shown that baicalein and its plasma metabolite baicalin do not only have antibacterial and antiviral activities, but also directly act on lymphocytes, macrophages, mast cells, neutrophils, and other immune cells to inhibit the production of biomarkers of the cytokine storm, such as IL-1β, IL-6, IFN-γ, and TNF-α, thus playing anti-inflammatory and immunomodulatory roles. [23][24][25][26][27][28][29][30][31] In addition, clinical studies have shown that high-dose oral administration of baicalein was safe and well tolerated. 32 Therefore, we put forward a conjecture: can baicalein and its metabolite baicalin be used in the treatment of cytokine storm caused by infection? However, baicalein itself is insoluble in water and slightly soluble in chloroform, and its oral bioavailability is low. It belongs to Class II under the Biopharmaceutical Classification System. At present, there is no commercially available active monomeric formulation of baicalein. Therefore, it is a great challenge to overcome multiple biological barriers and deliver baicalein to the lymphoid system where immune cells gather to play the function of anti-inflammatory and immune regulation.
A self-microemulsifying drug delivery system (SMEDDS), as a lipid carrier composed of an oil phase, emulsifier, and co-emulsifier, can be diluted and emulsified into an oil-in-water nanoemulsion by gastrointestinal fluid after oral administration. 33 Compared with other new delivery systems, SMEDDS can not only significantly increase the plasma concentration of insoluble drugs, but also significantly promote the lymphatic transport of drugs, and achieve targeted enrichment of drugs in mesenteric lymph nodes. 8 Therefore, in the past few years, self-emulsifying drug delivery systems have been widely used to improve the oral bioavailability of insoluble natural drugs and lymphoid absorption of immunomodulatory drugs, such as cyclosporine soft capsules (Novartis) and celecoxib oral solutions (Dr. Reddy's Laboratories). [34][35][36] In a previous study, we used a baicalein phospholipid complex as an intermediate carrier to construct a baicalein-phospholipid complex SMEDDS (BAPC-SME) for the first time. 37 Our study showed that BAPC-SME could significantly improve the oral bioavailability of baicalein. The C max of the BAPC-SME was 7.7 times and 1.9 times higher than those of the baicalein suspension (BA) and conventional baicalein SMEDDS (BA-SME), respectively. Also, the relative bioavailabilities of BAPC-SME were 448.7% and 131.0%, relative to BA and BA-SME, respectively. In addition, we also firstly studied the lymphatic transport of BAPC-SME. Our results showed that BAPC-SME could increase the lymphatic transport ratio of baicalein after oral administration from 18.8% for BA and 56.2% for BA-SME to 70.2%.
We suggested that the use of self-emulsifying nanocarriers for the encapsulation and delivery of baicalein could improve the transmembrane transport ability of drugs by promoting intestinal absorption and lymphatic transport, and promote the target delivery of drugs to mesenteric lymph nodes through the lymphatic transport pathway.
Thus, it is beneficial in the use of baicalein to regulate the inflammatory function of immune cells and is of breakthrough significance to enhance the treatment of cytokine storm with oral baicalein. Unfortunately, in our previous study, we did not investigate the effect of BAPC-SME on cytokine storm in vivo.
The existing studies on SMEDDS have focused on improving the solubility and bioavailability of insoluble drugs; however, there is a lack of in-depth and systematic research on lymph node targeting by SMEDDS and its improvement of the therapeutic management of cytokine storm. 38 The application of baicalein selfmicroemulsion in the treatment of cytokine storm has not been reported. Thus, based on the results of previous studies, the present study established mouse models of systemic cytokine storm and local lung cytokine storm to investigate the regulation of cytokine storm by BA, BA-SME, and BAPC-SME. The changes in the plasma concentrations of inflammatory cytokines among mice with systemic cytokine storm induced by lipopolysaccharide (LPS) were detected and analyzed for the first time. In addition, the present study also investigated the in vitro antibacterial and antiviral activities of BAPC-SME, the targeting of mesenteric lymph nodes by BAPC-SME, and explored the mechanism of uptake and transport of BAPC-SME by Caco-2 cells.

| Preparation and characterization of baicalein self-microemulsion based on phospholipid complexes
As described in a previous study, 37 BAPCs were prepared with BA and phospholipids in a mass ratio of 1:3.5 (wt/wt) using the solvent evaporation method. To prepare BAPC-SME, ethyl oleate, Tween 80, and Transcutol HP were weighed and mixed in a mass ratio of 2:5:3, after which BAPC was added. The mixture was shaken in an air bath oscillator overnight to obtain a transparent solution. BAPC-SME loaded with Cou-6 or DiR was obtained by dissolving fluorescent dye into the BAPC-SME.
According to the method of characterization of self-microemulsions described in a previous study, 37 the content, particle size, and zeta potential of the prepared BAPC-SME were simply characterized.

| Distribution of BAPC-SME in mesenteric lymph nodes
All rats were randomly divided into two groups and given DiR solution and DiR-labeled BAPC-SME at a dose of 1 mg/kg. The rats were sacrificed 0.5 and 2.0 h after administration, and their small intestines and mesenteric lymph nodes were excised. After washing with normal saline, the samples were subjected to fluorescence imaging in vitro using an IVIS Spectrum In Vivo Small Animal Imager (PerkinElmer).
Eighteen male Institute of Cancer Research mice were randomly divided into two groups. One group was given Cou-6 solution and the other was given Cou-6-labeled BAPC-SME intragastrically at a dose of 1 mg/kg. After 0.5, 1, and 2 h, the mice were sacrificed. Their mesenteric lymph nodes were excised and prepared into a single cell suspension using a cell sieve, after which their fluorescence intensity was detected using a C6 Flow Cytometer (BD Biosciences).

| Uptake of BAPC-SME by Caco-2 cells
Caco-2 cells seeded in 12-well plates were cultured at 37 C for 24 h, after which the cells were incubated with the solutions (100 ng/ml) of Cou-6 and Cou-6-labeled BAPC-SME in a constant temperature shaker at 37 C for 0.5, 1.0, and 1.5 h, respectively. Then, the cells were washed with phosphate-buffered saline (PBS) and digested with 0.25% trypsin. Finally, the cell suspension was analyzed using a C6 Flow Cytometer (BD Biosciences). In addition, Caco-2 cells seeded in a confocal dish were incubated with the solutions (100 ng/ml) of Cou-6 and Cou-6-labeled BAPC-SME in a shaker at a constant temperature of 37 C for 1 h. Another group of Caco-2 cells was pretreated at 4 C for 1 h, and then incubated with a pre-cooled Cou-6-labeled BAPC-SME solution at 4 C for 1 h. After incubation, the cells were washed with PBS, fixed with 4% paraformaldehyde and 0.5% Triton X-100, stained with TRITC phalloidin and DAPI, and finally observed using an FV1000 laser confocal microscope (Olympus).

| Cellular uptake and intracellular transport pathways of BAPC-SME in Caco-2 cells
To investigate the cellular uptake and intracellular transport pathways of BAPC-SME in Caco-2 cells, the cells were pretreated with methylβ-cyclodextrin, chlorpromazine hydrochloride, genistein, amiloride hydrochloride, brefeldin A, monensin, nocodazole, and bafilomycin A1 at 37 C for 1 h. Then, Cou6-labeled BAPC-SME solutions with different inhibitors at the same concentrations as described were added and incubated at 37 C for another 1 h. After incubation, all cells were treated and analyzed as previously described via flow cytometry and laser confocal microscopy.

| Lipopolysaccharide-induced systemic cytokine storm model
Male C57 mice were randomly divided into seven groups and fasted

| Inhibitory effect on systemic cytokine storm
To determine the effective dose of BAPC-SME, male C57 mice were randomly divided into seven groups and fasted for 12 h before the experiment. Both the blank control and model groups were given normal saline orally, while the experimental groups were given orally dif- and TNF-α were measured using the ELISA (Cat. no. E-EL-M0044c and 49c, Elabscience).

| Expression of inflammatory cytokines in mesenteric lymph nodes
The mesenteric lymph nodes were homogenized with Trizol, after which total intracellular RNA was extracted using an RNeasy Mini Kit.
Reverse transcriptional amplification was performed using an EvoM-MLV reverse transcription kit. The cDNA products obtained by reverse transcription were diluted and used as quantitative polymerase chain reaction (qPCR) templates to detect the expressions of the mRNA of TNF-α, IL-1β, IL-6, IL-12B, MCP-1, and TGF-β in mesenteric lymph nodes via quantitative real-time PCR assay.

| Western blot analysis
The mesenteric lymph nodes were mixed with radioimmunoprecipitation assay buffer containing protease and phosphatase inhibitors, and ground.
The supernatant was extracted as a protein sample after centrifugation

| Inhibitory effect on local cytokine storm in the lung
Male C57 mice were randomly divided into seven groups: the control, model, BA, BA-SME, BAPC-SME, and dexamethasone (positive drug) groups. First, they were given normal saline, different baicalein formulations (80 mg/kg), and dexamethasone (30 mg/kg). Then, LPS (5 mg/kg) was injected intratracheally to induce local cytokine storm in the lung after 40 min. The mice were sacrificed at 6 h after LPS injection, and the lung tissue was excised for hematoxylin and eosin (HE) staining, immunohistochemistry, and qPCR assay.

| Cytokine levels in lung tissue
Lung tissue was homogenized with Trizol, and total intracellular RNA was extracted using an RNeasy Mini Kit. Reverse transcriptional amplification was performed using an EvoM-MLV reverse transcription kit. The cDNA products obtained by reverse transcription were diluted and used as qPCR templates to detect the expression levels of the mRNA of TNF-α, MCP-1, IL-1β, IL-6, IL-10, and IFN-γ in lung tissue via quantitative real-time PCR assay.

| Histopathology
The lung tissue samples were fixed with 4% paraformaldehyde, embed-

| Immunohistochemistry
The lung tissue samples were fixed with 4% paraformaldehyde,

| Statistical analysis
All results are expressed as the mean ± standard error of the mean.
The statistical analyses were carried out using independent-samples t tests for two groups with SPSS version 17.0 (IBM Corporation), and one-way analysis of variance for multiple groups with GraphPad Prism V.7.00 for Windows (GraphPad Software). Differences were considered to be statistically significant at p < 0.05.

| Characterization of BAPC-SME
The results showed that the mean concentration of baicalein in BAPC-SME was 21.04 ± 0.25 mg/g. When BAPC-SME was mixed with water, the concentrate was completely emulsified in 1 min.
The average droplet size was 12.8 ± 0.2 nm with a polydispersity index of 0.08 ± 0.03, and zeta potential of À13.57 ± 0.38 mV ( Figure 2). Compared with the results of a previous study, 37 the particle size and zeta potential of BAPC-SME changed slightly, which may be related to the differences in suppliers of ethyl oleate and Tween 80.

| Distribution of BAPC-SME in mesenteric lymph nodes
In vitro fluorescence images of intestinal tracts and mesenteric lymph nodes of rats are shown in Figure 3b,c, respectively. The fluorescence intensity of mesenteric lymph nodes and intestinal tissues increased significantly after oral administration of DiR-labeled BAPC-SME for 0.5 h, which was significantly higher than that in the DiR solution group. The results suggested that BAPC-SME could promote the intestinal absorption of drugs and has a significant targeted aggregation in mesenteric lymph nodes.
We also quantitatively studied the distribution of Cou-

| Cellular uptake of BAPC-SME
In this study, Cou-6-labeled BAPC-SME was used for the quantitative analysis of cellular uptake. Flow cytometry was used to detect the fluorescence intensity of Caco-2 cells to characterize the cellular uptake of BAPC-SME. Cou-6-labeled BAPC-SME could be obviously absorbed by Caco-2 cells after incubation for 0.5, 1.0, and 1.5 h (Figure 4a,b) and the cellular uptake of Cou-6-labeled BAPC-SME was significantly higher than that of Cou-6 solution (p < 0.01). Simultaneously, the cellular uptake of Cou-6-labeled BAPC-SME increased gradually and reached saturation with increasing time, but did not increase after 1.0 h.
Further, DAPI and TRITC phalloidin were used to label the nucleus and cytoskeleton, respectively. A laser confocal microscope was used to observe the uptake of Cou-6-labeled BAPC-SME in Caco-2 cells at 37 C and 4 C. The cellular uptake of Cou-6-labeled BAPC-SME was significantly higher than that of Cou-6 solution at 37 C (Figure 4c). However, the cellular uptake of Cou-6-labeled BAPC-SME decreased significantly at 4 C. It is known that low temperatures lead to a decrease in the activity of various cellular enzymes, thus inhibiting the transmembrane transport of some substances that consume metabolic energy (adenosine triphosphate).
Therefore, we suspected that the cellular uptake of BAPC-SME depended on energy, and the transmembrane pathway might involve endocytosis.

| Cellular uptake and intracellular transport pathways of BAPC-SME
Previous studies have reported that methyl-β-cyclodextrin, chlorpromazine, genistein, and amiloride could inhibit cellular endocytosis mediated by lipid raft, clathrin, caveolin, and macropinocytosis, respectively. 39-44 After Caco-2 cells were incubated with Cou- In addition, after ingestion by Caco-2 cells, the microemulsion can be secreted into the tissue space through a series of intracellular transport pathways, thus entering the mesenteric lymphatic system.

F I G U R E 4
The cellular uptake of BAPC-SME in Caco-2 cells. (a,b) Quantification of the cellular uptake of Cou-6-labeled BAPC-SME and BA-SME at different times via flow cytometry (n = 3). (c) Confocal micrographs of the cellular uptake of Cou-6-labeled BAPC-SME in Caco-2 cells at 37 C and 4 C. The cellular uptake of Cou-6 solution at 37 C was set as the control.
However, when this process is inhibited, the microemulsion will remain in the cells. Some studies have shown that brefeldin A could block the transport pathway from the endoplasmic reticulum to the Golgi apparatus by triggering the retrograde transport of Golgi enzymes. Monensin inhibits the transport of macromolecules from the Golgi apparatus to the plasma membrane mainly by destroying the Golgi complex. 45 Nocodazole could bind to β-tubulin in microtubules to inhibit the formation of disulfide bonds and the dynamic changes in microtubules. It also destroys the structure of the Golgi apparatus and induces Golgi transport disorders. 46 Bafilomycin A1 can inhibit the maturation of early inclusions F I G U R E 5 The cellular uptake and intracellular transport pathways of BAPC-SME in Caco-2 cells. (a) Schematic diagram of cellular uptake and transport of BAPC-SME. The influence of different endocytosis inhibitors (b,c) and intracellular transport inhibitors (e,f) on the cellular uptake of Cou-6-labeled BAPC-SME was investigated via flow cytometry. All data were compared to those of Cou-6-labeled BAPC-SME without inhibitors (*p < 0.05, **p < 0.01, ***p < 0.001). The effects of methyl-β-cyclodextrin (d) and monensin (g) on the cellular uptake of Cou-6-labeled BAPC-SME were also observed via confocal laser scanning microscopy.
to lysosomes, thus interfering with the lysosome-mediated intracellular transport pathway of nanocarriers. 47 After Caco-2 cells were incubated with Cou-6-labeled BAPC-SME solution containing brefeldin A, monensin, nocodazole, and bafilomycin A1 for 1 h, the four inhibitors could significantly increase the concentration of Cou-6-labeled BAPC-SME in Caco-2 cells, among which monensin had the most obvious effect (Figure 5e-g). Compared with the control group without inhibitors, brefeldin A, monensin, nocodazole, and bafilomycin A1 increased the cellular uptake of Cou-6-labeled BAPC-SME by 45.7%, 178.2%, 88.3%, and 86.6%, respectively. The results suggested that the intracellular transport pathways of BAPC-SME in Caco-2 cells mainly included the endosomes, lysosomes, endoplasmic reticula, and Golgi apparatus.

| LPS-induced systemic cytokine storm model
The

| Inhibitory effect on systemic cytokine storm
The concentration of IL-6 in the plasma of mice increased by 122.7 times after intraperitoneal injection of LPS, and dexamethasone and 80 mg/kg of BAPC-SME could significantly inhibit the increase in the concentration of IL-6 ( Figure 7a). However, BAPC-SME showed no significant effect at doses of 20, 40, and 160 mg/kg. We speculated that the dose of 160 mg/kg was too high, so that BAPC-SME could not be completely emulsified, thus affecting the absorption of baicalein. Therefore, we chose 80 mg/kg as the effective dose of BAPC-SME and other baicalein formulations in vivo.

| Inhibitory effect on NF-κB and the STAT3 signal pathway
To explore the pharmacological mechanism by which BAPC-SME inhibits the expression of inflammatory cytokines in the mesenteric lymph nodes of mice in the systemic cytokine storm model, the expression levels of related proteins of the NF-κB and STAT3 signal pathway in mesenteric lymph nodes were determined via western blotting (Figure 8c). When compared with the model group, BAPC-SME was found to reduce the expression of NF-κB-p65 and STAT3 protein in lymph nodes, as well as inhibit the phosphorylation of NF-κB-p65, IκBα, and STAT3. Simultaneously, the inhibitory effect of BAPC-SME was stronger than that of BA and BA-SME. These results suggested that BAPC-SME could reduce the mRNA expression of downstream inflammatory cytokines by  (Table S4).
F I G U R E 7 Inhibitory effect of BAPC-SME on systemic cytokine storm. (a) Mean plasma concentration of IL-6 in C57BL/6 mice after oral administration of different doses of BAPC-SME following intraperitoneal injection of lipopolysaccharide (5 mg/kg, Table S5). (b) Mean plasma concentration of cytokine in C57BL/6 mice after oral administration of BA, BA-SME, and BAPC-SME following the intraperitoneal injection of lipopolysaccharide (Table S6). n = 4-6, *p < 0.05, **p < 0.01, ***p < 0.01 compared with model. The data of other cytokines are not shown in the picture.

| Inhibitory effect on local cytokine storm in the lung
Six hours after intratracheal injection of LPS, the mRNA expression of TNF-α, MCP-1, IL-1β, IL-6, and IL-10 in the lung tissue of mice was significantly increased (Figure 9b). Oral administration of BA and BA-SME The results further proved that BAPC-SME could target baicalein aggregation in mesenteric lymph nodes via lymphatic transport, which is beneficial in enabling baicalein to act directly on immune cells in lymph nodes to play an immunomodulatory role.
In addition, we speculated that BAPC-SME could not only promote the oral absorption of baicalein by increasing its solubility and dissolution, but also enhanced the absorption and lymphatic transport of baicalein through direct uptake by intestinal epithelial cells. Therefore, the uptake of BAPC-SME in intestinal epithelial cells was investigated using a Caco-2 cell model and its cellular uptake and the intracellular transport pathways involved were discussed. Based on the experimental study of this paper and the related literature, 49,50 the specific process of uptake and secretion of BAPC-SME by intesti- suggesting that BAPC-SME has the potential to regulate systemic cytokine storm induced by most bacterial and viral infections.

| Inhibitory effect of BAPC-SME on the NF-κB and STAT3 signal pathway
The NF-κB nuclear factor family is composed of c-Rel, Rel A (p65), Rel B, p50/p105, and p52/p100. In most inactivated cells, NF-κB binds to the inhibitor IκB in an inactive state. When cells are activated by extracellular stimuli such as pathogens, cytokines, and stress signals, IκB is phosphorylated, ubiquitinated, and degraded by protease, after which NF-κB is released and phosphorylated. Phosphorylated NF-κB is transferred to the nucleus to regulate a variety of targeted gene transcriptions in different cells, thus controlling the expression of proinflammatory cytokines, chemokines, immune receptors, and cell surface adhesion molecules. 55,56 Signal transducer and activator of transcription (STAT) can be phosphorylated and dimerized by Janus kinase (JAK) and transferred into the nucleus to regulate the expression of related genes; this signal pathway is called the JAK/STAT signal pathway. JAK/STAT protein is widely expressed and may play a role in regulating and maintaining a series of basic biological processes, including apoptosis, proliferation, immune response, and inflammation. 57,58 In the present study, western blotting showed that BAPC-SME could reduce the expression of NF-κB-p65 and STAT3 protein, and inhibit the phosphorylation of NF-κB-p65, IκBα, and STAT3 in the mesenteric lymph nodes of mice, thus blocking the activation of the NF-κB and STAT3 signal pathway and reducing the downstream mRNA expression of TNF-α, IL-1β, IL-6, IL-12B, and MCP-1. In some cases, persistent lung tissue damage without severe microbial infection is also associated with the clinical manifestations of cytokine storm. In addition to pulmonary infections, cytokine storm also results from severe infections in the gastrointestinal tract, urinary tract, central nervous system, skin, joint spaces, and other tissue.
In the present study, the therapeutic effects of different baicalein formulations on lung injury caused by LPS-induced cytokine storm were evaluated in vivo. The results showed that oral administration of BAPC-SME could significantly increase the inhibitory effect of baicalein on six kinds of cytokines in lung tissue: TNF-α, MCP-1, IL-1β, IL-6, IFN-γ, and IL-10. BAPC-SME also significantly improved pathological injury and T lymphocyte infiltration in lung tissue.

| CONCLUSIONS
In the present study, baicalein self-microemulsion with phospholipid complex as an intermediate carrier could significantly promote F I G U R E 9 Inhibitory effect of BAPC-SME on local cytokine storm in the lung. (a) The schematic of experimental process. (b) Relative mRNA level of TNF-α, MCP-1, IL-1β, IL-6, IL-10, and IFN-γ in the lung. The data are presented as the mean ± standard error of the mean (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 compared with model (Table S8)