The effect of prunella vulgaris extract on inflammatory response and peritoneal macrophages in septic mice

doi:10.11958/20240002

Abstract

Abstract:

Objective To investigate the effect of prunella vulgaris extract on inflammation, macrophage phenotype, and phagocytic ability in septic mice, and analyze whether Toll like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway involved in its mechanism. Methods C57BL/6 mice were divided into the control group, the model group and the prunella vulgaris extract low (25 mg/kg), medium (50 mg/kg) and high (100 mg/kg) dose groups. Except for the control group, all other groups of mice were injected intraperitoneally with lipopolysaccharide (LPS) to prepare sepsis model. Each group was given corresponding medication by gavage. After 24 hours of administration, serum tumor necrosis factor-α (TNF-α), interleukin (IL) -1β, high mobility group protein B1 (HMGB1), IL-10 levels, the proportion of M1 type (CD11b⁺F4/80⁺) and M2 type (CD206⁺F4/80⁺) macrophages in peritoneal macrophages, the phagocytotic capacity of macrophages, the expression of inducible nitric oxide synthase (iNOS) messenger RNA (mRNA) and arginase 1 (Arg1) mRNA in peritoneal macrophages and expression levels of TLR4, NF-κB p65 and their phosphorylated proteins in macrophages were detected. Results Compared with the control group, serum TNF-α, IL-1β, HMGB1, proportion of M1 type macrophages in abdominal cavity, mean fluorescence intensity and phagocytotic capacity of macrophages, iNOS mRNA, TLR4, phosphorylated NF-κB p65 (p-NF-κB p65)/NF-κB p65 protein expression were increased in the model group (P<0.05). IL-10, proportion of M2 type macrophages and Arg1 mRNA expression were decreased (P<0.05). Compared with the model group, serum TNF-α, IL-1β, HMGB1, proportion of M1 type macrophages in abdominal cavity, iNOS mRNA, TLR4, p-NF-κB p65/NF-κB p65 protein expression were decreased successively in the prunella vulgaris extract low, medium and high dose groups (P<0.05). IL-10, proportion of M2 macrophages, mean fluorescence intensity and phagocytotic capacity of macrophages and Arg1 mRNA expression were increased successively (P<0.05). Conclusion By inhibiting TLR4/NF-κB pathway, prunella vulgaris extract may inhibit the polarization of peritoneal macrophages into M1 type and promote their polarization to M2 type, enhance macrophage phagocytic ability and alleviate LPS induced inflammatory response in septic mice.

Key words: sepsis, macrophages, peritoneal, Prunellae Spica, Toll-like receptor 4, NF-kappa B, inflammation

CLC Number:

R515.3

Figures/Tables 10

References 23

[1]	YANG A, KENNEDY J N, REITZ K M, et al. Time to treatment and mortality for clinical sepsis subtypes[J]. Crit Care, 2023, 27(1):236. doi:10.1186/s13054-023-04507-5.
[2]	STANOJCIC M, VINAIK R, ABDULLAHI A, et al. NLRP3 knockout enhances immune infiltration and inflammatory responses and improves survival in a burn sepsis model[J]. Immunology, 2022, 165(2):195-205. doi:10.1111/imm.13427.
[3]	WANG J, LI J, LOU A, et al. Sacubitril/valsartan alleviates sepsis-induced acute lung injury via inhibiting GSDMD-dependent macrophage pyroptosis in mice[J]. FEBS J, 2023, 290(8):2180-2198. doi:10.1111/febs.16696.
[4]	LI J, YU S, LU X, et al. The phase changes of M1/M2 phenotype of microglia/macrophage following oxygen-induced retinopathy in mice[J]. Inflamm Res, 2021, 70(2):183-192. doi:10.1007/s00011-020-01427-w.
[5]	MENG Y, KONG K W, CHANG Y Q, et al. Histone methyltransferase SETD2 inhibits M1 macrophage polarization and glycolysis by suppressing HIF-1α in sepsis-induced acute lung injury[J]. Med Microbiol Immunol, 2023, 212(5):369-379. doi:10.1007/s00430-023-00778-5.
[6]	DI C, DU Y, ZHANG R, et al. Identification of autophagy-related genes and immune cell infiltration characteristics in sepsis via bioinformatic analysis[J]. J Thorac Dis, 2023, 15(4):1770-1784. doi:10.21037/jtd-23-312.
[7]	CHEN D, WANG H, CAI X. Curcumin interferes with sepsis-induced cardiomyocyte apoptosis via TLR1 inhibition[J]. Rev Port Cardiol, 2023, 42(3):209-221. doi:10.1016/j.repc.2023.01.013.
[8]	ZHAO H, WANG Y, ZHU X. Chrysophanol exerts a protective effect against sepsis-induced acute myocardial injury through modulating the microRNA-27b-3p/Peroxisomal proliferating-activated receptor gamma axis[J]. Bioengineered, 2022, 13(5):12673-12690. doi:10.1080/21655979.2022.2063560.
[9]	李露, 徐臣年, 杜燕, 等. 松果菊苷对脓毒症小鼠急性肾损伤的保护作用及其机制[J]. 山西医科大学学报, 2021, 52(4):456-462.
	LI L, XU C N, DU Y, et al. Protective effects and mechanism of echinacoside against sepsis-induced acute kidney injury[J]. J Shanxi Med Univ, 2021, 52(4):456-462. doi:10.13753/j.issn.1007-6611.2021.04.011.
[10]	LEI Y, YUAN H, GAI L, et al. Uncovering active ingredients and mechanisms of spica prunellae in the treatment of colon adenocarcinoma:a study based on network pharmacology and bioinformatics[J]. Comb Chem High Throughput Screen, 2021, 24(2):306-318. doi:10.2174/1386207323999200730210536.
[11]	JUN M S, KIM H S, KIM Y M, et al. Ethanol extract of Prunella vulgaris var. lilacina inhibits HMGB1 release by induction of heme oxygenase-1 in LPS-activated RAW 264.7 cells and CLP-induced septic mice[J]. Phytother Res, 2012, 26(4):605-612. doi:10.1002/ptr.3613.
[12]	何荷, 梁隆斌, 刘杨, 等. 绿原酸通过ROS/TXNIP/NLRP3信号通路介导的细胞焦亡途径减轻脓毒症小鼠急性肺损伤[J]. 中国病理生理杂志, 2021, 37(8):1455-1461.
	HE H, LIANG L B, LIU Y, et al. Chlorogenic acid attenuates acute lung injury in septic mice via ROS/TXNIP/NLRP3 signaling pathway mediated pyrocytosis pathway[J]. Chinese Journal of Pathophysiology, 2021, 37(8):1455-1461. doi:10.3969/j.issn.1000-4718.2021.08.015.
[13]	SONG J, ZHANG Z, HU Y, et al. An aqueous extract of Prunella vulgaris L. inhibits the growth of papillary thyroid carcinoma by inducing autophagy in vivo and in vitro[J]. Phytother Res, 2021, 35(5):2691-2702. doi:10.1002/ptr.7015.
[14]	GABARIN R S, LI M, ZIMMEL P A, et al. Intracellular and extracellular lipopolysaccharide signaling in sepsis:avenues for novel therapeutic strategies[J]. J Innate Immun, 2021, 13(6):323-332. doi:10.1159/000515740.
[15]	DING J, JIANG H, SU B, et al. DNMT1/miR-130a/ZEB1 regulatory pathway affects the inflammatory response in lipopolysaccharide-induced sepsis[J]. DNA Cell Biol, 2022, 41(5):479-486. doi:10.1089/dna.2021.1060.
[16]	CAI D, DUAN H, FU Y, et al. Renal tissue damage induced by acute kidney injury in sepsis rat model is inhibited by cynaropicrin via IL-1β and TNF-α down-regulation[J]. Dokl Biochem Biophys, 2021, 497(1):151-157. doi:10.1134/S1607672921020022.
[17]	CHEN J, WANG F, ZHANG S, et al. Activation of CD4⁺ T cell-derived cannabinoid receptor 2 signaling exacerbates sepsis via inhibiting IL-10[J]. J Immunol, 2022, 208(11):2515-2522. doi:10.4049/jimmunol.2101015.
[18]	SEOUDY W M, MOHY EL DIEN S M, ABDEL REHEEM T A, et al. Macrophages of the M1 and M2 types play a role in keloids pathogenesis[J]. Int Wound J, 2023, 20(1):38-45. doi:10.1111/iwj.13834.
[19]	TANG H, LIANG Y B, CHEN Z B, et al. Soluble egg antigen activates M2 macrophages via the STAT6 and PI3K pathways,and schistosoma japonicum alternatively activates macrophage polarization to improve the survival rate of septic mice[J]. J Cell Biochem, 2017, 118(12):4230-4239. doi:10.1002/jcb.26073.
[20]	CUI S, ZHANG Z, CHENG C, et al. Small extracellular vesicles from periodontal ligament stem cells primed by lipopolysaccharide regulate macrophage M1 polarization via miR-433-3p targeting TLR2/TLR4/NF-κB[J]. Inflammation, 2023, 46(5):1849-1858. doi:10.1007/s10753-023-01845-y.
[21]	NIU X, SONG H, XIAO X, et al. Tectoridin alleviates lipopolysaccharide-induced inflammation via inhibiting TLR4-NF-κB/NLRP3 signaling in vivo and in vitro[J]. Immunopharmacol Immunotoxicol, 2022, 44(5):641-655. doi:10.1080/08923973.2022.2073890.
[22]	WANG M, WEI J, SHANG F, et al. Down-regulation of lncRNA SNHG5 relieves sepsis-induced acute kidney injury by regulating the miR-374a-3p/TLR4/NF-κB pathway[J]. J Biochem, 2021, 169(5):575-583. doi:10.1093/jb/mvab008.
[23]	SENOUSY S R, AHMED A F, ABDELHAFEEZ D A, et al. Alpha-chymotrypsin protects against acute lung,kidney,and liver injuries and increases survival in CLP-induced sepsis in rats through inhibition of TLR4/NF-κB pathway[J]. Drug Des Devel Ther, 2022, 16:3023-3039. doi:10.2147/DDDT.S370460.

基因名称	引物序列（5′→3′）	产物大小/bp
iNOS	上游：GAACGGAGAACGTTGGATTTG 下游：TCAGGTCACTTTGGTAGGATTT	189
Arg1	上游：TTCCTGGCGATACCTCAGCAACC	252
Arg1	下游：CGCTAAGGCGAAAGCCCTCAAT	252
GAPDH	上游：GGTTGTCTCCTGCGACTTCA	114
GAPDH	下游：TGGTCCAGGGTTTCTTACTCC	114

基因名称	引物序列（5′→3′）	产物大小/bp
iNOS	上游：GAACGGAGAACGTTGGATTTG 下游：TCAGGTCACTTTGGTAGGATTT	189
Arg1	上游：TTCCTGGCGATACCTCAGCAACC	252
Arg1	下游：CGCTAAGGCGAAAGCCCTCAAT	252
GAPDH	上游：GGTTGTCTCCTGCGACTTCA	114
GAPDH	下游：TGGTCCAGGGTTTCTTACTCC	114

组别	n		TNF-α	IL-10
对照组	14		49.73±5.11	12.38±1.47
模型组	11		152.75±13.64^a	6.97±1.05^a
夏枯草提取物低剂量组	14		114.22±8.87^b	9.65±1.16^b
夏枯草提取物中剂量组	14		90.81±5.69^bc	12.19±1.23^bc
夏枯草提取物高剂量组	14		72.39±6.45^bcd	16.34±1.91^bcd
F			490.831^＊＊	78.441^＊＊
组别		IL-1β		HMGB1
对照组		68.06±7.79		108.74±12.15
模型组		172.14±13.23^a		584.56±47.24^a
夏枯草提取物低剂量组		139.37±11.06^b		420.04±36.05^b
夏枯草提取物中剂量组		111.91±10.42^bc		338.47±31.78^bc
夏枯草提取物高剂量组		81.66±9.54^bcd		212.65±30.69^bcd
F		207.901^＊＊		396.401^＊＊

组别	n		TNF-α	IL-10
对照组	14		49.73±5.11	12.38±1.47
模型组	11		152.75±13.64^a	6.97±1.05^a
夏枯草提取物低剂量组	14		114.22±8.87^b	9.65±1.16^b
夏枯草提取物中剂量组	14		90.81±5.69^bc	12.19±1.23^bc
夏枯草提取物高剂量组	14		72.39±6.45^bcd	16.34±1.91^bcd
F			490.831^＊＊	78.441^＊＊
组别		IL-1β		HMGB1
对照组		68.06±7.79		108.74±12.15
模型组		172.14±13.23^a		584.56±47.24^a
夏枯草提取物低剂量组		139.37±11.06^b		420.04±36.05^b
夏枯草提取物中剂量组		111.91±10.42^bc		338.47±31.78^bc
夏枯草提取物高剂量组		81.66±9.54^bcd		212.65±30.69^bcd
F		207.901^＊＊		396.401^＊＊

组别	n	M1型巨噬细胞比例	M2型巨噬细胞比例
对照组	14	6.52±0.64	20.66±2.01
模型组	11	30.97±2.18^a	3.42±0.37^a
夏枯草提取物低剂量组	14	24.55±2.07^b	8.57±0.64^b
夏枯草提取物中剂量组	14	17.28±1.65^bc	14.35±1.52^bc
夏枯草提取物高剂量组	14	10.94±1.32^bcd	25.98±2.13^bcd
F		463.581^＊＊	438.714^＊＊