生物活性透明质酸对脂多糖诱导的人树突状细胞和巨噬细胞炎症应答作用

doi:10.3877/cma.j.issn.1674-1358.2017.01.004

中华实验和临床感染病杂志(电子版) ›› 2017, Vol. 11 ›› Issue (01) : 14 -19. doi: 10.3877/cma.j.issn.1674-1358.2017.01.004

基础论著

生物活性透明质酸对脂多糖诱导的人树突状细胞和巨噬细胞炎症应答作用

尤娜¹, 褚萨萨¹, 朱进², 张馨³, 杨志国³, 汪茂荣⁴^,^†(

)

^1. 230032　合肥市，安徽医科大学附属解放军八一临床学院
^2. 210002　南京市，南京军区疾控中心流行病与微生物研究所
^3. 210002　南京市，解放军第81医院全军肝病中心
^4. 230032　合肥市，安徽医科大学附属解放军八一临床学院；210002　南京市，解放军第81医院全军肝病中心

收稿日期:2016-11-20 出版日期:2017-02-15
通信作者: 汪茂荣
基金资助:
"十二五"军队特需药品保密专项课题(No. 2013ZJ09J13110-05B)

Bioactive hyaluronic acid (B-HA) on lipopolysaccharide-induced inflammatory response in human dendritic cells and macrophages

Na You¹, Sasa Chu¹, Jin Zhu², Xin Zhang³, Zhiguo Yang³, Maorong Wang⁴^,^†()

^1. Department of Infectious Diseases, Anhui Medical University Affiliated Bayi Clinical College, Hefei 230032, China
^2. Department of Microbiology, Center of Diseases Control of Nanjing Military District, Nanjing 210002, China
^3. Institute of Liver Diseases, the 81st Hospital of PLA, Nanjing 210002, China
^4. Department of Infectious Diseases, Anhui Medical University Affiliated Bayi Clinical College, Hefei 230032, China; Institute of Liver Diseases, the 81st Hospital of PLA, Nanjing 210002, China

Received:2016-11-20 Published:2017-02-15
Corresponding author: Maorong Wang

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引用本文：

尤娜, 褚萨萨, 朱进, 张馨, 杨志国, 汪茂荣. 生物活性透明质酸对脂多糖诱导的人树突状细胞和巨噬细胞炎症应答作用[J/OL]. 中华实验和临床感染病杂志(电子版), 2017, 11(01): 14-19.

Na You, Sasa Chu, Jin Zhu, Xin Zhang, Zhiguo Yang, Maorong Wang. Bioactive hyaluronic acid (B-HA) on lipopolysaccharide-induced inflammatory response in human dendritic cells and macrophages[J/OL]. Chinese Journal of Experimental and Clinical Infectious Diseases(Electronic Edition), 2017, 11(01): 14-19.

目的

探讨经人透明质酸酶PH20特异处理的重组HA，即生物活性透明质酸（B-HA）对脂多糖（LPS）诱导的人树突状细胞（DC）和巨噬细胞炎症应答的影响。

方法

DC细胞和人急性单核细胞白血病单核细胞株（THP-1）经不同浓度的B-HA预先处理，加入LPS刺激不同时间，收集细胞和培养上清，采用实时荧光定量PCR和ELISA方法检测细胞因子mRNA和蛋白表达水平的变化。

结果

THP-1细胞实验中，与LPS组相比，B-HA 20 mg/ml不仅能够显著降低促炎因子肿瘤坏死因子-α（TNF-α）和白细胞介素-8（IL-8）的表达量，同时能够显著增加抗炎因子白细胞介素-10（IL-10）的表达量。其细胞中TNF-α、IL-8和IL-10 mRNA表达水平分别为24.41±1.94、35.76±4.19和4.99±0.07，而LPS组分别为38.48±1.47、62.7±1.93和2.27±0.16，差异均具有统计学意义（t = 5.781、P = 0.0290，t = 5.695，P = 0.0300，t = 15.20、P = 0.0040）。细胞上清中三者蛋白表达水平分别为（1 609.00±75.66）pg/ml、（213.10±9.14）pg/ml和（496.3±41.92）pg/ml，LPS组分别为（3 018±102.8）pg/ml、（587.4±3.140）pg/ml和（243.3±20.23）pg/ml，差异均具有统计学意义（t = 11.04、P = 0.0080，t = 38.74、P = 0.0007，t = 5.434、P = 0.0320）。在人外周血分化的DC细胞实验中，B-HA对细胞因子TNF-α、IL-8和IL-10的表达均无显著影响（P均> 0.05）。

结论

B-HA能有效调节人巨噬细胞炎性因子的表达，抑制其炎症反应；对人DC细胞的炎症反应无显著影响，提示B-HA对人炎性免疫细胞的炎症反应作用具有选择性。

关键词: 生物活性透明质酸, 脂多糖, 炎症反应, 树突状细胞, 人急性单核细胞白血病单核细胞株

Objective

To investigate the effects of hyaluronidase PH20 processed recombinant human hyaluronic acid fragments (bioactive HA, B-HA) on lipopolysaccharide (LPS)-induced inflammatory response in human dendritic cells (DC) and macrophages.

Methods

DC cells and human acute monocytic leukemia cells (THP-1) were pretreated with different concentrations of B-HA in absence or unabsence of LPS for different times. The levels of inflammatory cytokines in culture medium were measured by ELISA and inflammatory cytokines mRNA of cultured cells was detected by real-time PCR.

Results

Compared with LPS-only group, the expression of proinflammatory cytokines TNF-α and IL-8 were significantly inhibited by B-HA 20 mg/ml in THP-1 cells, while the expression of anti-inflammatory cytokine IL-10 was significantly elevated. The mRNA levels of TNF-α, IL-8 and IL-10 were 24.41±1.94, 35.76±4.19 and 4.99±0.07 in B-HA treated group, respectively, while the LPS-only group were 38.48±1.47, 62.7±1.93 and 2.27±0.16, with significant differences (t = 5.781, P = 0.0290; t = 5.695, P = 0.0300; t = 15.20, P = 0.0040). The TNF-α, IL-8 and IL-10 protein levels in culture medium were (1 609.00±75.66) pg/ml, (213.10±9.14) pg/ml and (496.30±41.92) pg/ml, while the LPS-only group were (3 018±102.8) pg/ml, ( 587.4±3.140) pg/ml and (243.3±20.23) pg/ml, with significant differences (t = 11.04, P = 0.0080; t = 38.74, P = 0.0007; t = 5.434, P = 0.0320). Unfortunately, no effect of B-HA was observed in human PBMC differentiated DC cells (all P > 0.05).

Conclusions

B-HA was effective on regulating inflammatory cytokines and could inhibit the inflammatory response in human macrophage. There was no significant effect on the inflammatory response in human PBMC-differentiated DC cells. The effect of B-HA on regulating inflammatory response is selective.

Key words: Bioactive hyaluronic acid, Lipopolysaccharide, Inflammatory response, Dendritic cells, Human acute monocytic leukemia cell line

表1 实时荧光定量PCR扩增引物序列

引物名称		引物序列（5′→3′）
GAPDH
	上游引物	GGCATTCACGAGACCACCTAC
	下游引物	CGACATGACGTTGTTGGCATAC
人TNF-α
	上游引物	CCTCTCTCTAATCAGCCCTCTG
	下游引物	GAGGACCTGGGAGTAGATGAG
人IL-8
	上游引物	ACTGAGAGTGATTGAGAGTGGAC
	下游引物	AACCCTCTGCACCCAGTTTTC
人IL-10
	上游引物	GACTTTAAGGGTTACCTGGGTTG
	下游引物	TCACATGCGCCTTGATGTCTG

表1 实时荧光定量PCR扩增引物序列

引物名称		引物序列（5′→3′）
GAPDH
	上游引物	GGCATTCACGAGACCACCTAC
	下游引物	CGACATGACGTTGTTGGCATAC
人TNF-α
	上游引物	CCTCTCTCTAATCAGCCCTCTG
	下游引物	GAGGACCTGGGAGTAGATGAG
人IL-8
	上游引物	ACTGAGAGTGATTGAGAGTGGAC
	下游引物	AACCCTCTGCACCCAGTTTTC
人IL-10
	上游引物	GACTTTAAGGGTTACCTGGGTTG
	下游引物	TCACATGCGCCTTGATGTCTG

表2 THP-1细胞中各组细胞因子mRNA的表达量（±s）

组别	TNF-α	IL-8	IL-10
空白组	1.11±0.08	1.33±0.20	0.99±0.00
LPS组	38.48±1.47	62.07±1.93	2.27±0.16
LPS + B-HA 5 mg/ml组	35.74±2.86	56.87±4.52	3.28±0.05
LPS + B-HA 10 mg/ml组	33.57±1.37	56.35±3.63	3.77±0.14
LPS + B-HA 20 mg/ml组	24.41±1.94	35.76±4.19	4.99±0.07
t值	5.7810	5.6950	15.2300
P值	0.0290	0.0300	0.0040

表2 THP-1细胞中各组细胞因子mRNA的表达量（±s）

组别	TNF-α	IL-8	IL-10
空白组	1.11±0.08	1.33±0.20	0.99±0.00
LPS组	38.48±1.47	62.07±1.93	2.27±0.16
LPS + B-HA 5 mg/ml组	35.74±2.86	56.87±4.52	3.28±0.05
LPS + B-HA 10 mg/ml组	33.57±1.37	56.35±3.63	3.77±0.14
LPS + B-HA 20 mg/ml组	24.41±1.94	35.76±4.19	4.99±0.07
t值	5.7810	5.6950	15.2300
P值	0.0290	0.0300	0.0040

表3 DC中各组细胞因子mRNA的表达量（±s）

组别	TNF-α	IL-8	IL-10
空白组	0.93±0.06	0.94±0.04	0.94±0.05
LPS组	192.00±0.66	34.42±2.85	2.41±0.14
LPS + B-HA 5 mg/ml组	257.70±32.36	38.98±2.80	1.92±0.30
LPS + B-HA 10 mg/ml组	143.80±10.89	32.64±1.20	2.60±0.28
LPS + B-HA 20 mg/ml组	183.00±6.98	27.63±1.82	3.00±0.09
t值	1.9060	1.3590	1.6860
P值	0.1970	0.3071	0.1904

表3 DC中各组细胞因子mRNA的表达量（±s）

组别	TNF-α	IL-8	IL-10
空白组	0.93±0.06	0.94±0.04	0.94±0.05
LPS组	192.00±0.66	34.42±2.85	2.41±0.14
LPS + B-HA 5 mg/ml组	257.70±32.36	38.98±2.80	1.92±0.30
LPS + B-HA 10 mg/ml组	143.80±10.89	32.64±1.20	2.60±0.28
LPS + B-HA 20 mg/ml组	183.00±6.98	27.63±1.82	3.00±0.09
t值	1.9060	1.3590	1.6860
P值	0.1970	0.3071	0.1904

图1 B-HA 20 mg/ml在不同时间点对LPS诱导的THP-1细胞中细胞因子mRNA的影响

图2 B-HA对LPS诱导的THP-1细胞培养上清中细胞因子蛋白表达水平的影响

1	Hill DR, Rho HK, Kessler SP, et al. Human milk hyaluronan enhances innate defense of the intestinal epithelium[J]. J Biol Chem,2013,288(40):29090-29104.
2	Liang J, Jiang D, Noble PW. Hyaluronan as a therapeutic target in human disease[J]. Adv Drug Deliv Rev,2016,97:186-203.
3	Jiang D, Liang J, Noble PW. Hyaluronan as an immune regulator in human diseases[J]. Physiol Rev,2011,91(1):221-264.
4	Walicová V, Gajdziok J, Gajdošová M, et al. The influence of hyaluronan addition on thickness, weight, uniformity of mass and water content of mucoadhesive films[J]. Ceska Slov Farm,2016,65(3):94-98.
5	Campo GM, Avenoso A, Campo S, et al. Small hyaluronan oligosaccharides induce inflammation by engaging both toll-like-4 and CD44 receptors in human chondrocytes[J]. Biochem Pharmacol,2010,80(4):480-490.
6	Noble PW. Hyaluronan and its catabolic products in tissue injury and repair[J]. Matrix Biol,2002,21(1):25-29.
7	Wang Y, Han G, Guo B, et al. Hyaluronan oligosaccharides promote diabetic wound healing by increasing angiogenesis[J]. Pharmacol Rep,2016,68(6):1126-1132.
8	赵含丹，惠觅宙. 生物活性透明质酸片段治疗慢性咽炎初步探讨[J]. 临床军医杂志,2014,42(8):864-865.
9	陈丹，王小东，童静植, 等. 三种分离人外周血单核细胞方法的比较[J]. 天津医科大学学报,2014,20(6):483-485.
10	姚康，葛均波，孙爱军, 等. 高糖对人单核细胞源树突状细胞分化成熟和免疫功能的影响及其机制研究[J]. 中华心血管病杂志,2006,34(1):60-64.
11	Park EK, Jung HS, Yang HI, et al. Optimized THP-1 differentiation is required for the detection of responses to weak stimuli[J]. Inflamm Res,2007,56(1):45-50.
12	Chen GY, Nunez G. Sterile inflammation: sensing and reacting to damage[J]. Nat Rev Immunol,2010,10(12):826-837.
13	Kolar SL, Kyme P, Tseng CW, et al. Group B Streptococcus evades host immunity by degrading hyaluronan[J]. Cell Host Microbe,2015,18(6):694-704.
14	Taylor KR, Yamasaki K, Radek KA, et al. Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2[J]. J Biol Chem,2007,282(25):18265-18275.
15	Huang Z, Zhao C, Chen Y, et al. Recombinant human hyaluronidase PH20 does not stimulate an acute inflammatory response and inhibits lipopolysaccharide-induced neutrophil recruitment in the air pouch model of inflammation[J]. J Immunol,2014,192(11):5285-5295.
16	Dong Y, Arif A, Olsson M, et al. Endotoxin free hyaluronan andhyaluronan fragments do notstimulate TNF-α, interleukin-12or upregulate co-stimulatorymolecules in dendritic cells ormacrophages[J]. Sci Rep,2016,6:36928.
17	Wakao N, Imagama S, Zhang H, et al. Hyaluronan ligosaccharides promote functional recovery after spinal cord injury in rats[J]. Neur Lett,2011,488(3):299-304.
18	Yamasaki K, Muto J, Taylor KR, et al. NLRP3/cryopyrin is necessary for interleukin-1beta (IL-1beta) release in response to hyaluronan, an endogenous trigger of inflammation in response to injury[J]. J Biol Chem,2009,284(19):12762-12771.
19	Xu C, Chen G, Yang W, et al. Hyaluronan ameliorates LPS-induced acute lung injury in mice via Toll-like receptor (TLR) 4-dependent signaling pathways[J]. Int Immunopharmacol,2015,28(2):1050-1058.
20	Taylor KR, Yamasaki K, Radek KA, et al. Recognition of hyaluronan released in sterile injury involves a unique receptor complex dependent on Toll-like receptor 4, CD44, and MD-2[J]. J Biol Chem,2007,282(25):18265-18275.
21	Tao L, Ding Q, Gao C, et al. Resveratrol attenuates neuropathic pain through balancing pro-inflammatory and anti-inflammatory cytokines release in mice[J]. Int Immunopharmacol,2016,34:165-172.
22	Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells[J]. Nat Rev Immunol,2010,10(3):170-181.
23	Ray A, Dittel BN. Mechanisms of regulatory B cell function in autoimmune and inflammatory diseases beyond IL-10[J]. J Clin Med,2017,6(1):12.
24	Moore KW, de Waal Malefyt R, Coffman RL, et al. Interleukin-10 and the interleukin-10 receptor[J]. Annu Rev Immunol,2001,19:683-765.
25	Muto J, Morioka Y, Yamasaki K, et al. Hyaluronan digestion controls DC migration from the skin [J]. J Clin Invest,2014,124(3):1309-1319.

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