Objective:

2

To investigate the effects of astragaloside Ⅳ (AS-Ⅳ) on podocyte injury of diabetic nephropathy (DN) and reveal its potential mechanism.

Methods:

2

In

in vitro

experiment

podocytes were divided into 4 groups

normal

high glucose (HG)

inositol-requiring enzyme 1 (IRE-1) α activator (HG+thapsigargin 1 μmol/L)

and IRE-1α inhibitor (HG+STF-083010

20 μmol/L) groups. Additionally

podocytes were divided into 4 grou

ps

including normal

HG

AS-Ⅳ (HG+AS-Ⅳ 20 μmol/L)

and IRE-1α inhibitor (HG+STF-083010

20 μmol/L) groups

respectively. After 24 h treatment

the morphology of podocytes and endoplasmic reticulum (ER) was observed by electron microscopy. The expressions of glucose-regulated protein 78 (GRP78) and IRE-1α were detected by cellular immunofluorescence. In

in vivo

experiment

DN rat model was established via a consecutive 3-day intraperitoneal streptozotocin (STZ) injections. A total of 40 rats were assigned into the normal

DN

AS-Ⅳ [AS-Ⅳ 40 mg/(kg•d)

]

and IRE-1α inhibitor [STF-083010

10 mg/(kg•d)

]

groups (

n

=10)

respectively. The general condition

24-h urine volume

random blood glucose

urinary protein excretion rate (UAER)

blood urea nitrogen (BUN)

and serum creatinine (SCr) levels of rats were measured after 8 weeks of intervention. Pathological changes in the renal tissue were observed by hematoxylin and eosin (HE) staining. Quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to detect the expressions of GRP78

IRE-1α

nuclear factor kappa Bp65 (NF-κBp65)

interleukin (IL)-1β

NLR family pyrin domain containing 3 (NLRP3)

caspase-1

gasdermin D-N (GSDMD-N)

and nephrin at the mRNA and protein levels

in vivo

and

in vitro

respectively.

Results:

2

Cytoplasmic vacuolation and ER swelling were observed in the HG and IRE-1α activator groups. Podocyte morphology and ER expansion were improved in AS-Ⅳ and IRE-1α inhibitor groups compared with HG group. Cellular immunofluorescence showed that compared with the normal group

the fluorescence intensity of GRP78 and IRE-1α in the HG and IRE-1α activator groups were significantly increased whereas decreased in AS-Ⅳ and IRE-1α inhibitor groups (

P

<

0.05). Compared with the normal group

the mRNA and protein expressions of GRP78

IRE-1α

NF-κBp65

IL-1β

NLRP3

caspase-1 and GSDMD-N in the HG group was increased

(

P

<

0.05). Compared with HG group

the expression of above indices was decreased in the AS-Ⅳ and IRE-1α inhibitor groups

and the expression in the IRE-1α activator group was increased (

P

<

0.05). The expression of nephrin was decreased in the HG group

and increased in AS-Ⅳ and IRE-1α inhibitor groups (

P

<

0.05). The

in vivo

experiment results revealed that compared to the normal group

the levels of blood glucose

triglyceride

total cholesterol

BUN

SCr and urinary protein in the DN group were higher (

P

<

0.05). Compared with DN group

the above indices in AS-Ⅳ and IRE-1α inhibitor groups were decreased (

P

<

0.05). HE staining revealed glomerular hypertrophy

mesangial widening and mesangial cell proliferation in the renal tissue of the DN group. Compared with the DN group

the above pathological changes in renal tissue of AS-Ⅳ and IRE-1α inhibitor groups were alleviated. Quantitative RT-PCR and Western blot results of GRP78

IRE-1α

NF-κBp65

IL-1β

NLRP3

caspase-1 and GSDMD-N were consistent with immunofluorescence analysis.

Conclusion:

2

AS-Ⅳ could reduce ERS and inflammation

improve podocyte pyroptosis

thus exerting a podocyteprotective effect in DN

through regulating IRE-1α/NF-κB/NLRP3 signaling pathway.