Inflammation Aggravates Intracellular Cholesterol Accumulation and Injury in HMCs Loaded by Low-Density Lipoprotein via Up-Regulating NPC1 Expression

Li Qianqian¹, Yang Xuejun¹, Yang Haiping¹, Chen Yaxi², Zhang Gaofu¹*, Li Qiu¹*

(¹Department of Nephrology, Children’s Hospital of Chongqing Medical University, Key Laboratory of the Ministry of Education, Key Laboratory of Pediatrics in Chongqing, Chongqing International Sciene and Technology Cooperation Cebter for Child Development and Disorders, Chongqing Key Laboratory of Child Infection and Immunity, Chongqing 400014, China; ²Key Laboratory of Metabolism on Lipid and Glucose, Center for Lipid Research, Chongqing Medical University, Chongqing 400014, China)

Abstract:

Investigate the effect of IL1-β on NPC1 (Niemann-pick protein c1) expression in human mesangial cell (HMCs) loaded by low-density lipoprotein (LDL) and explore whether NPC1-mediated cholesterol accumulation causing cell injury. HMCs were divided into control group, LDL group, LDL+IL-1β group. The level of NPC1 protein was detected by Western blot. The total cholesterol and endoplasmic reticulum cholesterol were detected by a cholesterol detection kit. Cell proliferation was measured using a cell count kit. Cell cycle was detected by flow cytometry PI/RN staining. The mRNA levels of NPC1, glucose-regulated protein78(GRP78), protein kinase R-like endoplasmic reticulum kinase (PERK), activating transcription factor 6 (ATF6), fibronectin (FN), type IV collagen secretion (Col IV) were measured by real-time quantitative PCR. GRP78 and FN protein level were detected using immunofluorescent staining. After using U-18666A to intervene the function of NPC1, the level of endoplasmic reticulum cholesterol, cell proliferation, cell cycle, GRP78, FN and ColIV mRNA was measured. We found that LDL loading alone significantly increased the expression of NPC1, promoted intracellular and endoplasmic reticulum cholesterol accumulation, accelerated the proliferation of HMCs, increased the ratio of S phase in cell cycle, and promoted the expression of endoplasmic reticulum stress marker (GRP78, PERK, ATF6 mRNA) and mesangial matrix marker (FN, Col IV mRNA), increased the mean fluorescence intensity of GRP78 and FN. Inflammation further aggravated the level of the above indicators in HMCs loaded by LDL. Compared with LDL+IL-1β group, co-treatment with U-18666A significantly decreased the level of endoplasmic reticulum cholesterol, cell proliferation, ratio of S phase in cell cycle and GRP78, FN, Col IV mRNA. These results suggested that inflammation aggravated intracellular and endoplasmic reticulum cholesterol accumulation in HMCs via up-regulating NPC1 expression and caused endoplasmic reticulum stress (ERS) and cell injury.

CSTR: 32200.14.cjcb.2019.06.0008