Objective

2

To explore the protective mechanism of

Tongqiao Mingmu IV Formula

（通窍明目Ⅳ号方） on glaucoma-induced optic nerve damage.

Methods

2

Tongqiao Mingmu IV Formula

was divided into

Shugan Formula

（疏肝方） and

Mingmu Formula

（明目方）. Thirty rats were randomly assigned to whole formula group， Shugan

group， Mingmu

group， normal group， and vitamin B

12

group， with five rats in each group. The whole formula group received 94.5 g/（kg·d） of

Tongqiao Mingmu IV Formula

by gavage； Shugan group received 40.5 g/（kg·d） of

Shugan Formula

by gavage； Mingmu

group received 54.0 g/（kg·d） of

Mingmu Formula

by gavage； the control group received 18.9 ml/（kg·d） of distilled water by gavage； and the vitamin B

12

group received 50 μg/（kg·d） of vitamin B

12

by gavage. Administer oral gavage once daily for 7 consecutive days， then collect blood from the abdominal aorta and isolate serum. R28 retinal precursor cell lines were divided into blank group， model group， normal serum group， whole formula serum group， Sh

ugan serum group， Mingmu serum group， and vitamin B

12

serum group， with three replicate wells per group. Except for the blank group， all other groups employed L-glutamate to induce R28 cell damage using optimized induction concentrations and durations. After modeling， the blank group and model group received no serum intervention； the normal serum group had its medium removed and replaced with 10% normal serum； the whole formula serum group， Shugan serum group， and Mingmu serum group had their medium removed and replaced with 10% serum from the corresponding herbal formula group； the vitamin B

12

serum group had its medium removed and replaced with 10% vitamin B

12

serum. After 48 hours of continued culture， cell viability including survival rate and inhibition rate was assessed using the CCK-8 assay； apoptosis rates were determined via PI/Hoechst dual staining， comparing mean fluorescence intensities； reactive oxygen species （ROS） levels were measured using fluorescent probes； immunofluorescence， Western Blot， and qRT-PCR were used to detect the expression of proteins and mRNA related to the NOD-like receptor protein 3 （NLRP3）/Caspase-1/IL-1β signaling pathway.

Results

2

Cells were treated with 20 mmol/L L-glutamate for 48 hours to induce a cellular injury model. Compared with the blank group， the model group exhibited decreased R28 cell viability， increased cell inhibition rate， elevated average fluorescence intensity of PI/Hoechst staining， and increased levels of cellular ROS， NLRP3/Caspase-1/IL-1β-related proteins， and their mRNA expression （

P

＜0.05）. Compared with the model group， all serum intervention groups showed varying degrees of improvement in the aforementioned indicators （

P

＜0.05）. The whole formula serum group demonstrated superior effectiveness to the vitamin B

12

serum group in reducing inhibition rates， mean PI/Hoechst staining fluorescence intensity， ROS levels， and mRNA expression of NLRP3/Ca

spase-1/IL-1β-related proteins （

P

＜0.05）. The mean PI/Hoechst staining fluorescence intensity in whole formula serum group and Shugan serum group was lower than that in the Mingmu serum group （

P

＜0.05）.

NLRP3

，

Apoptosis

-

Related Speck

-

Like Protein

（

ASC

），

Caspase

-

1

， and

IL

-

1β

mRNA in the whole formula serum group was lower than that in both Shugan serum group and Mingmu serum group （

P

＜0.05）.

Conclusion

2

Tongqiao Mingmu IV Formula

may alleviate programmed cell necrosis by inhibiting the NLRP3/Caspase-1/IL-1β signaling pathway， thereby effectively reducing retinal neuronal apoptosis and achieving optic nerve protection.