Objective

2

To observe the effect of the serum containing Puerariae Lobatae Radix on tumor necrosis factor-

α

（TNF-

α

）， endoplasmic reticulum stress signaling pathway protein endoplasmic reticulum stress protein glucose-regulated protein 78 （GRP78）， activated transcription factor 4 （ATF4）， protein kinase R-like endoplasmic reticulum kinase （PERK）， and eukaryotic translation initiation factors （eIF2

α

） and on inflammatory injury of macrophages induced by high glucose， and explore its possible mechanism.

Method

2

The control serum and serum containing Puerariae Lobatae Radix were prepared by serum pharmacology. The RAW264.7 macrophages were cultured

in vitro

， and 62.5 mmol·L

-1

glucose was used to induce macrophages to establish a model of severe endoplasmic reticulum （ER） stress （ERS）

in vitro

. Different volume fractions of serum containing Puerariae Lobatae Radix and ERS inhibitor （4-PBA） were used to interfere with the cells. Different glucose concentrations （22.5， 23.5， 25.0， 27.5， 32.5， 47.5，

72.5， 122.5 mmol·L

-1

） and the effect of different volume fractions of serum containing Puerariae Lobatae Radix （0%， 2.5%， 5%， 7.5%， 10%， 15%， 20%） on the survival rate of RAW264.7 macrophages were detected by cell proliferation and cell counting kit-8 （CCK-8） assay. Based on the results of the effect of glucose concentrations on macrophage survival rate by CCK-8， Western blot （WB） was used to determine the protein expression levels of GRP78， the signature protein of ERS， by different concentrations of glucose （22.5， 32.5， 42.5， 52.5， 62.5， 72.5 mmol·L

-1

） at different time periods （6， 12， 24， 36， 48 h）， and the optimal concentration and time for establishing the model of severe ERS were screened out. Based on the above experimental results， the cells were divided into blank group， 62.5 mmol·L

-1

glucose model group， 2 mmol·L

-1

4-PBA group， and high， medium， and low-dose （15%， 10%， 5%） serum containing Puerariae Lobatae Radix groups for subsequent experiments. The expression of TNF-

α

in the supernatant of RAW264.7 macrophages in the model of severe ERS was determined by enzyme-linked immunosorbent assay （ELISA）. The expressions of related pathway proteins in the model of severe ERS were determined by WB.

Result

2

The results of CCK-8 assay showed that the survival rate of macrophages reached the highest under the stimulation of glucose concentration of 27.5 mmol·L

-1

（

P

<

0.01）， while the survival rate of macrophages increased with the concentration increasing from 22.5 mmol·L

-1

to 27.5 mmol·L

-1

. When the glucose concentration was 25.0 mmol·L

-1

， there was a significant difference （

P

<

0.01）， and when the glucose concentration was 37.5 mmol·L

-1

to 122.5 mmol·L

-1

， there was a downward trend. The serum containing Puerariae Lobatae Radix showed significant differences in the volume of 1% to 15% （

P

<

0.05，

P

<

0.01）. The results of WB found that the GRP78 protein expression was the most significant at 24 h （

P

<

0.01）， and the GRP78 protein expression was the most significant when the glucose concentration was 62.5 mmol·L

-1

（

P

<

0.01）. Therefore， 62.5 mmol·L

-1

of glucose was the optimal concentration to induce the model of severe ERS. As compared with the blank group， the protein expression levels of TNF-

α

， GRP78， ATF4， phosphorylation（p）-PERK/PERK， p-eIF2

α

/eIF2

α

in the model group were significantly increased （

P

<

0.05，

P

<

0.01）， and as compared with the model group， the protein expression levels of TNF-

α

， GRP78， ATF4， p-PERK/PERK， p-eIF2

α

/eIF2

α

in each administration group were significantly decreased （

P

<

0.05，

P

<

0.01）.

Conclusion

2

The results of

in vitro

experiments show that Puerariae Lobatae Radix can alleviate the inflammatory injury of macrophages induced by high glucose to a certain extent and restore cell homeostasis by inhibiting the expression of GRP78， ATF4， PERK， and eIF2

α

in the ERS signaling pathway.