EGCG promotes PRKCA expression to alleviate LPS-induced acute lung injury and inflammatory response

Acute lung injury (ALI), which could be induced by multiple factors such as lipopolysaccharide (LPS), refer to clinical symptoms of acute respiratory failure, commonly with high morbidity and mortality. Reportedly, active ingredients from green tea have anti-inflammatory and anticancer properties, including epigallocatechin-3-gallate (EGCG). In the present study, protein kinase C alpha (PRKCA) is involved in EGCG protection against LPS-induced inflammation and ALI. EGCG treatment attenuated LPS-stimulated ALI in mice as manifested as improved lung injury scores, decreased total cell amounts, neutrophil amounts and macrophage amounts, inhibited the activity of MPO, decreased wet-to-dry weight ratio of lung tissues, and inhibited release of inflammatory cytokines TNF-α, IL-1β, and IL-6. PRKCA mRNA and protein expression showed to be dramatically decreased by LPS treatment while reversed by EGCG treatment. Within LPS-stimulated ALI mice, PRKCA silencing further aggravated, while PRKCA overexpression attenuated LPS-stimulated inflammation and ALI through MAPK signaling pathway. PRKCA silencing attenuated EGCG protection. Within LPS-induced RAW 264.7 macrophages, EGCG could induce PRKCA expression. Single EGCG treatment or Lv-PRKCA infection attenuated LPS-induced increases in inflammatory factors; PRKCA silencing could reverse the suppressive effects of EGCG upon LPS-stimulated inflammatory factor release. In conclusion, EGCG pretreatment inhibits LPS-induced ALI in mice. The protective mechanism might be associated with the inhibitory effects of PRKCA on proinflammatory cytokine release via macrophages and MAPK signaling pathway.


Materials and methods
Animal. Male BALB/c mice (pathogen free; 16-20 g of weight) were obtained from Hunan SJA Laboratory Animal Co., Ltd (Changsha, China) and raised in a pathogen free condition. The mice were kept in temperaturecontrolled rooms in a 12:12 h light-dark cycle and provided with food and water at will. Before the study, the animals underwent at least seven days of adaptation. All procedures involving animals have been approved by the Animal Experiment Ethics Committee of Central South University. All procedures were conducted based on recommendations in guidelines for the care and use of laboratory animals. All procedures were performed using the 30 mg/kg doses of 2% pentobarbital sodium by intraperitoneal injection anesthesia and every effort was made to minimize the pain for mice.

LPS-induced acute lung injury mouse model.
A total of 40 male BALB/c mice were randomly divided into 4 groups: the control group in which mice only received sterile saline treatment, the EGCG group in which mice only received EGCG treatment, the LPS group in which mice only received LPS stimulation, and the LPS + EGCG group in which mice received LPS stimulation and EGCG treatment. For LPS stimulation, each mouse was anesthetized with pentobarbital sodium by intraperitoneal injection and intratracheally injected by LPS (5 mg/Kg b.wt) 27 . The mice were then placed vertically and rotated for 1 min to distribute the drops to the lungs. For EGCG treatment, mice were administered EGCG (10 mg/kg b.wt, intraperitoneal injection) 1 h prior to LPS injection. The administration and dose of EGCG were selected according to a previous study 28 . Mice were euthanized to collect study samples at 24 h after the intratracheal administration of LPS.
For PRKCA silencing or overexpression, each mouse was injected through the caudal vein with 2 × 10 7 transducing units (TU) of Lv-shPRKCA or Lv-PRKCA (Lv-NC used as a negative control; Genetop, Changsha, China) lentivirus. After 5 days, mice were treated or non-treated with EGCG and intratracheally injected with LPS as described above.

Bronchoalveolar lavage fluid (BALF) and cells counting.
After anesthetizing and sacrificing the mice, the lungs of the mice were lavaged with 1.0 mL PBS (pH 7.2) 3 times, the upper trachea was intubated, and the BALF of the mice was collected. The BALF was centrifuged at 700 × g for 5 min below 4 °C. The pelleted cells were resuspended in 50 μL of PBS and stained with Diff-Quik (International Reagents Corp., Kobe, Tokyo, Japan) to prepare for cell counting. Then, cell numbers were double-blind counted, including total cells, neutrophils, macrophages and lymphocytes with a hemocytometer and Giemsa staining 29,30 . Histopathologic examination and lung injury evaluation. Lung tissues were collected and fixed in 10% formalin, embedded in paraffin, sliced into 5-μm thick sections, and stained with hematoxylin and eosin (H&E) for the detection of the pathological changes in the lung tissues. Finally, histopathology was observed under an optical microscope (DSX100, Olympus, Tokyo, Japan). Lung injury score was measured according to the methods reported previously 31,32 . The criteria are as follows: score 0 = no damage, score l = mild damage, score 2 = moderate damage, score 3 = severe damage, score 4 = very severe histologic changes. ELISA analysis. BALF  For LPS stimulation, cells were activated by incubation in a medium containing LPS (100 ng/ml) when the cells grew to a density of 2 × 10 6 cells/ml.
Isolation of alveolar macrophages. The isolation and characterization of murine alveolar macrophages were perform based on previous research 33,34 . In brief, BALF pellet from all mice groups was seeded in Petri dishes with RPMI 1640 Medium supplemented with 10% FBS, 100 IU/mL penicillin and 100 µg/mL streptomycin (Gibco BRL, Grand Island, USA), for 1 h at 37 °C. The supernatant was discarded and purified attached alveolar macrophages were cryopreserved in 500 µL of TRIzol reagent (Sigma-Aldrich, St. Louis, USA). Alveolar macrophages' purity was assessed by Diff-Quick staining and immunofluorescence.
Polymerase chain reaction (PCR)-related analysis. Total RNA was isolated from tissue samples by Trizol reagent (Invitrogen). The PCR-related analyses were performed using a reaction system described before 35 for 35 cycles. The mouse GAPDH housekeeping gene was used as an internal control. The relative expression levels were calculated using the 2 −ΔΔCT method.
Ethics approval and consent to participate. All procedures performed in studies involving animals were in accordance with the ethical standards of Central South University and with the 1975 Helsinki declaration and ARRIVE guidelines.

Results
EGCG alleviates LPS-stimulated ALI within mouse model. To confirm the specific roles of EGCG in LPS-induced ALI, the study established the murine model of LPS-stimulated ALI. We randomly divided the mice into four groups: the control group, the EGCG group, the LPS group, and the LPS + EGCG group, which received corresponding treatment. H&E staining on mice lung showed that, in comparison with the control and single EGCG group, single LPS treatment induced lung injury, as manifested as inflammatory cell infiltration, edema and hemorrhage and subdued necrosis, whereas EGCG treatment on LPS-stimulated mice alleviated LPS-induced lung injury (Fig. 1A). The results of H&E staining were further evidenced by lung injury score evaluation that LPS stimulation dramatically increased the lung injury score, which was observably decreased by about 30% by EGCG co-treatment when compared to LPS group (P < 0.05, Fig. 1B). Consistently, LPS stimulation induced sharp increases in total cell number and neutrophil number and a significant increase in macrophage number, whereas caused no change in lymphocyte number when compared to control group (P < 0.01, Fig. 1C); LPS-induced increases in total cell, neutrophil, and macrophage numbers were significantly attenu- www.nature.com/scientificreports/ ated by EGCG co-treatment (P < 0.05, Fig. 1C). Moreover, LPS stimulation significantly enhanced MPO activity (P < 0.01, Fig. 1D) and increased lung wet/dry weight ratio (P < 0.01, Fig. 1E), which could both be reduced by about 40% and 30% respectively by EGCG co-treatment when compared to LPS group (P < 0.05, Fig. 1D,E). These data suggest that LPS induces ALI in mice lung, while EGCG treatment attenuates ALI in mice induced by LPS.

EGCG decreases the levels of the inflammatory cytokines in vivo.
To further validate the inflammation in mice lung in each group, the expression levels of inflammatory factors, including TNF-α, IL-6, IL-1β, in BALF were examined. Figure 2 showed that when compared with control group, LPS treatment dramatically increased TNF-α, IL-6, and IL-1β levels (P < 0.001); while compared to LPS group, EGCG decreased the levels of TNF-α, IL-6, and IL-1β by about 30%, 40% and 35% respectively (P < 0.01). In summary, LPS stimulation induces inflammation reaction within mice lung, which could be attenuated by EGCG treatment.

EGCG modulates PRKCA expression within LPS-mediated ALI mice.
To further investigate the mechanism of EGCG improving LPS-induce ALI, we screened for proteins that interact with EGCG and proteins with top ten interaction scores were shown in Table 1.
Mice were divided into four groups as mentioned above and the expression of the top ten genes predicted to interact with EGCG showed to be examined within the control and the LPS group. As shown in Fig. 3A, compare Mice were randomly divided into four groups: the control group, the EGCG group, the LPS group, and the LPS + EGCG group. Mice in each group were treated accordingly and examined for (A) the pathological changes in lung tissues by hematoxylin and eosin (H&E) staining (200 ×). scale bar = 50 μm; (B) lung injury scores following the methods reported before; (C) bronchoalveolar lavage fluid (BALF) cell numbers including total cells, neutrophils, macrophages and lymphocytes using a hemocytometer and Giemsa staining; (D) MPO activity using a spectrophotometer; (E) lung wet/dry weight ration. **P < 0.01 compared to Control group; #P < 0.05 compared to LPS group.  Figure 3. EGCG modulates PRKCA expression in LPS-induced ALI mice. Mice were divided into four groups (the control group, the EGCG group, the LPS group, and the LPS + EGCG group) accordingly. (A) The expression levels of the top ten genes predicted to interact with EGCG were examined in the control group and the LPS group using real-time qPCR. (B) PRKCA mRNA expression in mouse lung tissues from each group was determined using real-time qPCR. (C) PRKCA protein levels in mouse lung tissues from each group were determined using Immunoblotting. *P < 0.05, **P < 0.01 compared to Control group; ##P < 0.01 compared to LPS group. www.nature.com/scientificreports/ to control group, LPS stimulation significantly inhibited PRKCA and PRKCB expression by about 70% and 30%, respectively (P < 0.05). But, as displayed in Fig. S1, LPS stimulation observably restrained PRKCB mRNA (P < 0.05, Fig. S1A) and protein (P < 0.05, Fig. S1B) expression; while EGCG had no significant effect on the mRNA and protein expression of PRKCB. And, in mice lung, LPS stimulation significantly reduced by about 65% when compared with control group; while EGCG treatment reversed the mRNA expression (P < 0.01, Fig. 3B) and the protein levels (P < 0.01, Fig. 3C) of PRKCA, in comparison with the control group and single LPS group. Moreover, PRKCA might be involved in H 2 O 2 -induced injury to A549 cells 24 ; H 2 O 2 exposure decreased protein kinase C (PKC)-α activity 24 . Thus, we chose PRKCA for further experiments. These data further suggest that PRKCA might participate in the protective effects of EGCG against LPS-mediated ALI in mice.

PRKCA effects upon LPS-mediated ALI.
To validate the specific roles of PRKCA in EGCG protection against LPS-mediated ALI, we randomly divided the mice into seven groups: the control group, the LPS group, the LPS + EGCG group, the LPS + Lv-NC group, the LPS + Lv-shPRKCA group, the LPS + Lv-PRKCA group, and the LPS + EGCG + Lv-shPRKCA group, which received treatment and/or injection accordingly. In lung tissues of mice from the LPS, LPS + Lv-NC, and LPS + Lv-shPRKCA groups, PRKCA mRNA and protein expression showed to be dramatically decreased than those in the control group (P < 0.01, Fig. 4A). Upon LPS stimulation, EGCG treatment and Lv-PRKCA infection significantly increased PRKCA mRNA expression and protein levels, and EGCG treatment also relieved the effect of PRKCA silencing on PRKCA mRNA expression and protein levels (Fig. 4A). In macrophages of alveolar, when compared to control group, PRKCA expression level was markedly decreased in the LPS group (P < 0.05, Fig. 4B). Upon LPS stimulation, EGCG treatment and Lv-PRKCA infection notably increased PRKCA expression level; knockdown of PRKCA further inhibited PRKCA expression level; and EGCG treatment reversed the inhibitory effect of PRKCA silencing on PRKCA expression level (P < 0.05, Fig. 4B). As for lung injury evaluation, H&E staining and lung injury scores indicated that PRKCA silencing further aggravated LPS-mediated pulmonary injury (Fig. 4C,D), EGCG treatment and PRCKA overexpression improved LPS-mediated pulmonary injury (Fig. 4C,D), and EGCG treatment-induced improvement of LPS-induced lung injury was significantly abolished by PRKCA silencing (Fig. 4C,D). Moreover, LPS-induced increases in total cell amounts, neutrophil amounts, and macrophage amounts were further increased by PRKCA silencing while decreased by EGCG treatment and PRKCA overexpression; PRKCA silencing attenuated EGCG effects upon LPS-mediated increases in cell numbers (Fig. 4E). Moreover, the activity of MPO and the wet-todry weight ratio of lung issues showed the similar trend, that is, LPS significantly increased, whereas PRKCA silencing further increased the activity of MPO and the wet/dry ratio; LPS-induced increases were reduced via EGCG treatment and PRKCA overexpression (Fig. 4F,G). EGCG treatment attenuated LPS-mediated elevations in these indexes, while PRKCA silencing attenuated the effects of EGCG treatment (Fig. 4F,G). These data suggest that PRKCA silencing further aggravates LPS-induced lung injury and attenuates EGCG protection against LPS-mediated pulmonary injury.

PRKCA effects upon the levels of the inflammatory cytokines in vivo through MAPK signaling pathway.
Regarding the inflammatory microenvironment in LPS-mediated ALI mice, we examined the BALF levels of inflammatory factors (TNF-α, IL-6, and IL-1β) within each group. LPS stimulation significantly enhanced TNF-α, IL-6, and IL-1β levels (P < 0.01); PRKCA silencing further increased, while EGCG treatment and PRKCA overexpression decreased LPS-mediated increases within the levels of TNF-α, IL-6, and IL-1β (Fig. 5A). EGCG reduced TNF-α, IL-6, and IL-1β levels upon LPS stimulation, and EGCG effects were significantly reversed by PRKCA silencing (P < 0.01, Fig. 5A). According to the KEGG website (https:// www. kegg. jp/ kegg-bin/ show_ pathw ay? ko040 10+ K02677) 36 , protein kinase C (PKC) family is located upstream of MAPK signaling pathway, affecting a variety of physiological processes through the MAPK signaling pathway. Moreover, previous studies had shown that MAPK signaling pathway is involved in the process of lung injury [37][38][39] . Hence, we hypothesized that PRKCA alleviated acute lung injury in mice by regulating MAPK signaling pathway. Then, we detected the expression levels of MAPK signaling pathway-related proteins (p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK) in each group (Fig. 5B). LPS stimulation observably enhanced p-p38, p-ERK1/2, and p-JNK protein levels about 210%, 220% and 180% respectively when compared with control group (P < 0.01); knockdown of PRKCA further increased, while EGCG treatment and overexpression of PRKCA notably decreased LPS-mediated increases within p-p38, p-ERK1/2, and p-JNK protein levels. However, when compared with LPS + Lv-NC group, the inhibitory effect of EGCG on p-p38, p-ERK1/2, and p-JNK protein levels couldn't observed in LPS + EGCG + Lv-shPRKCA group; the protective effect of EGCG might be eliminated by the effect of silencing of PRKCA (P < 0.01, Fig. 5B). In summary, PRKCA might participate in the protective effects of EGCG against LPS-stimulated pulmonary injury through modulating inflammatory factor release and MAPK signaling pathway.

Effects of PRKCA on LPS-induced inflammatory responses through MAPK pathway in vitro.
To investigate the specific role of PRKCA in inflammatory factor release, the study treated mouse macrophage RAW 264.7 cells in different ways. We divided RAW 264.7 cells into seven groups: the negative control group, the LPS group, the LPS + EGCG group, the LPS + Lv-NC group, the LPS + Lv-shPRKCA group, the LPS + Lv-PRKCA group, and the LPS + EGCG + Lv-shPRKCA group. Cells in each group were treated and/or infected accordingly. As observed in tissue samples, PRKCA protein level was significantly decreased by about 60% by LPS stimulation compared to control group (P < 0.01, Fig. 6A,B). Under LPS stimulation, when compared with LPS group, EGCG or Lv-PRKCA infection restored, while Lv-shPRKCA further decreased PRKCA protein level; the effects of EGCG on PRKCA protein level were reversed by Lv-shPRKCA infection (Fig. 6A,B).  (E) cell numbers including total cells, neutrophils, macrophages and lymphocytes using a hemocytometer; (F) MPO activity using a spectrophotometer; (G) lung wet/dry weight ration. *P < 0.05, **P < 0.01, compared to the control group. #P < 0.05, ##P < 0.01, compared to the LPS + Lv-NC group. As for inflammatory factors, LPS promoted IL-6, IL-1β, and TNF-α protein contents. Single EGCG treatment or Lv-PRKCA infection attenuated LPS-induced increases in inflammatory factors (Fig. 6A,C-E), while Lv-shPRKCA infection further increased LPS-induced increases in these factors (Fig. 6A,C-E). The effects of EGCG on inflammatory factors were partially reversed by Lv-shPRKCA infection (Fig. 6A,C-E).
As for MAPK signaling pathway-related proteins (p-p38, p38, p-ERK1/2, ERK1/2, p-JNK, JNK), LPS notably facilitated p-p38, p-ERK1/2, and p-JNK protein contents. Single EGCG treatment or Lv-PRKCA infection relieved LPS-induced increases in MAPK signaling pathway-related proteins, while overexpression of PRKCA further promoted LPS-induced increases in these proteins. The inhibitory effects of EGCG on MAPK signaling pathway-related proteins were partially reversed by knockdown of PRKCA (Fig. 6F). These data suggest that PRKCA silencing could reverse the inhibitory effects of EGCG on inflammatory factor release through regulating MAPK signaling pathway.

Discussion
Herein, this study revealed that PRKCA is involved in EGCG protection against LPS-induced inflammation and ALI. CALI is a frequent complication following sepsis in critically ill patients and LPS is thought to be the most important pathogen that leads to the development of ALI in sepsis 40 . As previously reported, LPS-stimulated ALI is related to elevated infiltration of inflammatory cells and significant pulmonary damage, with the major characteristic features of the alterations within histopathological characteristics and other markers 40,41 . In the www.nature.com/scientificreports/ present study, both H&E staining and lung injury scoring showed that LPS stimulation caused significantly lung injury to mice lung, whereas EGCG treatment improved LPS-induced histopathological changes and lung injury. The pathological process of LPS-stimulated ALI involves the TLR4/NF-κB pathway, which initiates intracellular inflammatory signal transduction 42,43 . Following TLR4 treatment and LPS treatment, MyD88 pathway can transport signals to TNF receptor-associated factor-6 (TRAF6), resulting in IκB degradation and NF-κB activation 42,43 . The nucleus-translocation of NF-κB results in increases in proinflammatory transmitters 44 , thereby amplifying a cascade of inflammation, promoting the transfer of neutrophils into alveoli and damaging pulmonary tissues 42,43 . Myeloperoxidase (MPO) activity was applied to assess the activation and accumulation of neutrophils and polymorphonuclear leukocyte in the lung tissues 45,46 . Herein, LPS indeed elevated the total cell number, the neutrophil numbers, and the macrophage number in the BALF, and the MPO activity, while EGCG treatment significantly attenuated these changes induced by LPS, indicating that EGCG treatment alleviates LPS-triggered inflammatory cascade.
Gram-negative bacterial infections are the major cause of ALI 28 . The recognition of LPS that has entered the body is achieved through specialized receptors known as pattern recognition receptors (PPRs), which plays a role in inflammatory signal transduction, leading to the synthesis and release of pro-inflammatory cytokines, such as TNF-α, IL-1β, and IL-6. The imbalance between pro-inflammatory and anti-inflammatory transmitters is a vital event during ALI development 9,10 . The macrophages and endothelial cells stimulated with TNF-α produce substantial amounts of proinflammatory transmitters, including IL-1β and IL-8, and secondary inflammatory mediators, including platelet activator and NO 47 . IL-1β contribute to promoting tissue cell sensitivity to TNF-α and stimulating other inflammatory cytokines, including TNF-α, IL-8, E-selectin, and P-selectin. IL-6 induces acute inflammation, enhances neutrophil recruitment and activation, leads to lung edema, and causes systemic inflammation reaction 9,47 . These three pro-inflammatory cytokines of TNF-α, IL-1β, and IL-6 enhance each other and trigger a cascade of inflammation response resulting in ALI. In the present study, EGCG significantly reduced LPS-mediated elevations within the levels of TNF-α, IL-1β, and IL-6, further confirming EGCG protection against LPS-mediated lung injury via improving the cascade of inflammation reaction.
As for the mechanism underlying the protective effects of EGCG, herein, we revealed that PRKCA expression, which is predicted to interact with EGCG, could be significantly downregulated by LPS stimulation. The PRKCA has been reported to mediate many physiological activities and the role of PRKCA in lung injury may be controversial. Liu et al. elucidated that the PKC signaling pathway were activated by mechanical ventilation in ventilator-induced lung injury 48 . The activation of protein kinase C participated in lung injury in endotoxemic sheep 49 . However, PRKCA may also plays a protective role in lung injury and impairs inflammatory response. The inflammatory responses in the airways are related to the generation of reactive oxygen species (ROS), including H 2 O 2 and superoxide (O 2 -), leading to widespread pulmonary damage within respiratory illness; notably, H 2 O 2 exposure decreased PRKCA activity by causing translocation of PRKCA from the membrane to the cytoplasm 24 . Moreover, Lin et al. illuminated that up-regulation of PYK2/PRKCA-dependent haem oxygenase-1 attenuates TNF-α-induced lung inflammation 50 . In the present study, LPS significantly downregulated, whereas EGCG treatment significantly reversed PRKCA mRNA expression and protein levels, suggesting that PRKCA might participate in EGCG protection against LPS-mediated pulmonary damage. As expected, PRKCA silencing further aggravated, whereas PRKCA overexpression attenuated LPS-mediated pulmonary damage via improving histopathological alterations, reducing cell numbers, inhibiting the activity of MPO, and reducing proinflammatory cytokine levels. More importantly, after silencing PRKCA, the protective effects of EGCG were significantly attenuated, indicating that PRKCA participates in EGCG protection against LPS-induced lung injury.
Alveolar macrophages are the main resident phagocytes in lung, which participate in the host's initial defense response through producing inflammatory mediators and chemokines, and regulate the initiation and development of pulmonary inflammation 51,52 . Meanwhile, excessive activation of macrophages is also the key mechanism of inflammatory damage. TNF-α and IL-1β are the key components of cytokine network and the important inflammation mediators of ALI initiation, which mainly secreted by mononuclear macrophages 53,54 . To further address the molecular mechanism underlying PRKCA-mediated EGCG protection against LPS-mediated ALI, we examined the specific effects of PRKCA and the dynamic effects of EGCG and PRKCA on LPS-stimulated mouse macrophage RAW264.7 cells. Consistent with previous studies, within LPS-stimulated RAW264.7 cells, EGCG treatment increased PRKCA protein and reduced proinflammatory cytokine levels; after silencing PRKCA within LPS-induced RAW264.7 cells, EGCG inhibition upon proinflammatory cytokines showed to be significantly attenuated. In summary, PRKCA participates in EGCG protection against LPS-stimulated pulmonary damage through affecting proinflammatory cytokine release by macrophages.
Taken together, the present study revealed that EGCG pretreatment inhibits LPS-induced ALI in mice. The protective mechanism might be associated with the inhibitory effects of PRKCA on proinflammatory cytokine release via macrophages.

Data availability
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