Cancer-associated fibroblasts (CAFs), which have the properties of myofibroblasts, are fibroblasts in tumor tissue that are activated by cancer cells and. CAFs can provide nutritional support and invasion channels for tumor cells, thereby helping tumor cell proliferation, invasion and metastasis. This paper observed the effect of Liujunzi Decoction (六君子汤) on the apoptosis of esophageal cancer-related CAFs and discussed its molecular mechanism, so as to provide experimental basis for the clinical application of Liujunzi Decoction in the treatment of esophageal cancer.

①The effect of TE-1 conditioned medium (TE-1M) on CAFs activity was detected by MTT method. ②The effect of Liujunzi Decoction on CAFs activity was detected by MTT method. ③The effects of Liujunzi Decoction on the secretion of transforming growth factor-β1 (TGF-β1) from CAFs were measured by ELISA. ④Flow cytometry was performed to detect the effect of Liujunzi Decoction on apoptosis of CAFs. ⑤The effects of Liujunzi Decoction on mRNA expression levels of CAFs apoptosis-related molecules were detected by RT-qPCR.6. The effects of Liujunzi Decoction on apoptosis-related molecular protein expression levels of CAFs were detected by Western Blot.

①The results of MTT showed that compared with the blank group, 50 μg/mL Liujunzi Decoction had no significant effect on the activity inhibition of CAFs, and the other concentration groups had an inhibitory effect on the activity of CAFs (P<0.05). ②The results of MTT showed that compared with the blank group, 10%, 20% and 30% TE-1M had a promoting effect on the proliferation of CAFs, among which 20% TE-1M had the most significant proliferation effect on CAFs (P<0.001), and 40% TE-1M (P<0.05) and 50% TE-1M (P<0.01) had a significant inhibitory effect on the proliferation of CAFs. ③The results of ELISA method showed that compared with the blank group, the content of TGF-β1 secreted by CAFs in the model group decreased (P<0.001), the content of TGF-β1 in the Liujunzi Decoction group was significantly increased (P<0.001), and the content of TGF-β1 in the SB-431542 inhibitor+Liujunzi Decoction group was decreased compared with that in the Liujunzi Decoction group (P<0.001). ④The results of flow cytometry showed that there was no significant difference in the apoptosis rate in the model group compared with that in the blank group. Compared with the model group, the apoptosis rate of the Liujunzi Decoction group was significantly increased (P<0.001), and the apoptosis rate of the SB-431542 inhibitor+Liujunzi Decoction group was significantly decreased compared with the Liujunzi Decoction group (P<0.001). ⑤The results of PCR showed that the expressions of Smad2 and FAP genes in the model group were increased (P<0.01, P<0.001) compared with those in the blank group, and the expressions of Smad2, Caspase-3 and FAP in the Liujunzi Decoction group were significantly increased (P<0.05, P<0.01, P<0.001) compared with those in the model group. Compared with those of the Liujunzi Decoction group, the expressions of Smad2, Caspase-3 and FAP in the were significantly decreased (P<0.05, P<0.001). ⑥The results of Western blot showed that compared with the blank group, the expression of Smad2 protein in the model group decreased (P<0.01). The expressions of Smad2 and Caspase-3 in the Liujunzi Decoction group were significantly increased (P<0.001) compared with those in the model group and the expression level of FAP protein was increased (P<0.05). Compared with those in the Liujunzi Decoction group, the expressions of Smad2 and Caspase-3 were significantly decreased in the SB-431542 inhibitor+Liujunzi Decoction group (P<0.01), and the expression of FAP protein was decreased (P<0.05).

The proliferation of CAFs was promoted by inhibiting TGF-β1/Smad2 pathway in indirect coculture with esophageal cancer cells. Liujunzi Decoction can induce CAFs apoptosis through TGF-β1/Smad2 pathway, and the induction effect of TGF-β1/Smad2 pathway on apoptosis of CAFs is more obvious in co-culture environment.