Exploring the treatment of sepsis-associated acute lung injury with Liangge Powder via ERK1/2 and PI3K/AKT pathways: based on network pharmacology and whole animal experimentation / 中华劳动卫生职业病杂志

Objective: To investigate the therapeutic effect and mechanism of Liangge Powder against sepsis-induced acute lung injury (ALI) . Methods: From April to December 2021, the key components of Liangge Powder and its targets against sepsis-induced ALI were analyzed by network pharmacology, and to enrich for relevant signaling pathways. A total of 90 male Sprague-Dawley rats were randomly assigned to sham-operated group, sepsis-induced ALI model group (model group), Liangge Powder low, medium and high dose group, ten rats in the sham-operated group and 20 rats in each of the remaining four groups. Sepsis-induced ALI model was established by cecal ligation and puncture. Sham-operated group: gavage with 2 ml saline and no surgical treatment. Model group: surgery was performed and 2 ml saline was gavaged. Liangge Powder low, medium and high dose groups: surgery and gavage of Liangge Powder 3.9, 7.8 and 15.6 g/kg, respectively. To measure the wet/dry mass ratio of rats lung tissue and evaluate the permeability of alveolar capillary barrier. Lung tissue were stained with hematoxylin and eosin for histomorphological analysis. The levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL) -6 and IL-1β in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay. The relative protein expression levels of p-phosphatidylinositol 3-kinase (PI3K), p-protein kinase B (AKT), and p-ertracellular regulated protein kinases (ERK) were detected via Western blot analysis. Results: Network pharmacology analysis indicated that 177 active compounds of Liangge Powder were selected. A total of 88 potential targets of Liangge Powder on sepsis-induced ALI were identified. 354 GO terms of Liangge Powder on sepsis-induced ALI and 108 pathways were identified using GO and KEGG analysis. PI3K/AKT signaling pathway was recognized to play an important role for Liangge Powder against sepsis-induced ALI. Compared with the sham-operated group, the lung tissue wet/dry weight ratio of rats in the model group (6.35±0.95) was increased (P<0.001). HE staining showed the destruction of normal structure of lung tissue. The levels of IL-6 [ (392.36±66.83) pg/ml], IL-1β [ (137.11±26.83) pg/ml] and TNF-α [ (238.34±59.36) pg/ml] were increased in the BALF (P<0.001, =0.001, <0.001), and the expression levels of p-PI3K, p-AKT and p-ERK1/2 proteins (1.04±0.15, 0.51±0.04, 2.31±0.41) were increased in lung tissue (P=0.002, 0.003, 0.005). The lung histopathological changes were reduced in each dose group of Liangge Powder compared with the model group. Compared with the model group, the wet/dry weight ratio of lung tissue (4.29±1.26) was reduced in the Liangge Powder medium dose group (P=0.019). TNF-α level [ (147.85±39.05) pg/ml] was reduced (P=0.022), and the relative protein expression levels of p-PI3K (0.37±0.18) and p-ERK1/2 (1.36±0.07) were reduced (P=0.008, 0.017). The wet/dry weight ratio of lung tissue (4.16±0.66) was reduced in the high-dose group (P=0.003). Levels of IL-6, IL-1β and TNF-α[ (187.98±53.28) pg/ml, (92.45±25.39) pg/ml, (129.77±55.94) pg/ml] were reduced (P=0.001, 0.027, 0.018), and relative protein expression levels of p-PI3K, p-AKT and p-ERK1/2 (0.65±0.05, 0.31±0.08, 1.30±0.12) were reduced (P=0.013, 0.018, 0.015) . Conclusion: Liangge Powder has therapeutic effects in rats with sepsis-induced ALI, and the mechanism may be related to the inhibition of ERK1/2 and PI3K/AKT pathway activation in lung tissue.

The impact of modified Liangge powder on platelet activation markers and release of proinflammatory cytokine of mice by stimulation of lipopolysaccharide / 中国中西医结合急救杂志

Objective To observe the impact of modified Liangge powder (MLP) on platelet activation markers and the release of proinflammatory cytokine in mice by stimulation of lipopolysaccharide (LPS). Methods 112 male mice were randomly divided into control group, model group and MLP low, middle and high dose treatment groups. The sepsis model was reproduced by injection of LPS 10 mg/kg into a mouse tail vein. In the control group, normal saline 10 mg/kg was injected into the tail vein of mouse. The MLP low, middle, and high dose groups received 0.94, 1.89, 2.84 g/mL MLP 0.02 mL/g by gavage respectively for 3 days, while the control group and model group received equal amount of normal saline by gavage for 3 days. After modeling for 24 hours and 72 hours, 8 mice in each of the three different dose MLP groups and model group were killed and their blood was taken. In the control group, after modeling for 24 hours, 8 mice were killed and their blood was taken. Platelet (PLT) was counted by blood cell analyzer, plasma interleukin-10 (IL-10), high mobility group protein B1 (HMGB1) and platelet factor 4 (PF4) were detected by enzyme-linked immunosorbent assay (ELISA). In each group after modeling for 72 hours, another 8 mice were taken, and laser scanning confocal microscopy was used to measure the platelet cytosolic Ca2+ concentration. Results Compared with the control group, the level of PLT at 24 hours(×109/L: 347.70±115.10 vs. 1 013.10±136.60) was decreased, and the levels of IL-10 (μg/L: 356.86±34.72 vs. 39.50±23.45), HMGB1 (mg/L: 16.24±4.49 vs. 10.75±1.91), PF4 (μg/L: 5.43±0.61 vs. 1.33±0.40) and Ca2+ (nmoL/L: 8.60±0.52 vs. 1.05±0.33) were elevated in model group. Compared with the model group, the levels of PLT in the MLP high, middle and low dose groups were all significantly elevated; the increase in PLT in middle dose group after modeling for 72 hours was the most remarkable (×109/L:952.13±104.02 vs. 771.50±129.30, P < 0.05); the levels of IL-10, HMGB1, PF4, Ca2+ in MLP low, middle, high dose groups were significantly decreased. The most obvious degree of decrease in level of the following indexes were as follows:IL-10 in MLP high dose group at 72 hours after modeling (μg/L:110.17±29.12 vs. 441.50±30.72), HMGB1 in MLP high dose group after modeling for 24 hours (mg/L: 10.33±3.52 vs. 16.24±4.49), PF4 in MLP middle dose group after modeling for 24 hours (μg/L:2.08±0.92 vs. 5.43±0.61) and Ca2+ in MLP high dose group (nmoL/L:2.97±0.96 vs. 8.60±0.52, all P<0.05). Conclusion MLP may possibly down-regulate the inflammatory cytokines release induced by LPS to inhibit the activation of platelet Ca2+, in turn prevent the activation of platelet and improve thrombocytopenia caused by LPS.