Based on Toll-like receptor 5 (TLR5)/nuclear factor-kappa B (NF-κB) signaling pathway and gut microbiota, the mechanism of different doses of Xianglian Pills (香连丸) on Helicobacter pylori-associated gastritis (HPAG) rats with damp-heat in spleen-stomach syndrome was explored.

Sixty SPF healthy male Wistar rats were fed adaptively for one week and divided into blank group (10 rats) and intervention group (50 rats) by random number table method. The intervention group utilized a multi-factor modeling approach to construct a rat model of HPAG with damp-heat in spleen-stomach syndrome. After successful modeling, the intervention group was further divided into model group, Xianglian Pills low-dose group (0.54 g·kg^-1), Xianglian Pills medium-dose group (1.08 g·kg^-1), Xianglian Pills high-dose group (2.16 g·kg^-1) and western medicine group, with 10 rats in each group. Blank group and model group were given the equal volume of distilled water for 21 consecutive days. During the observation period, the body weight of the rats was measured weekly. After the administration, the fresh feces of the rats were collected and 16S rRNA sequencing technology was used to investigate the differences and changes in gut microbiota among the groups. Hematoxylin-eosin (HE) staining was employed to examine the histopathological changes of gastric mucosa in the rats. Giemsa staining was used to detect Helicobacter pylori in gastric mucosa of the rats. The serum levels of tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) in the rats were measured by enzyme-linked immunosorbent assay (ELISA). Western blot was performed to determine the protein levels of TLR5, TNF receptor-related factor 6 (TRAF6), transforming growth factor β-activated kinase 1 (TAK1), nuclear factor κB inhibitor kinase α(IKKα), NF-κB, myeloid differentiation factor 88 (MyD88), NF-κB p50 protein and NF-κB p65 protein in the rats.

Compared with those in the blank group, the average body weight of the rats in the model group decreased significantly (P<0.01), and the symptoms of damp-heat in spleen-stomach syndrome were obvious (P<0.01). The gastric mucosal epithelial cells exhibited disordered arrangement, with partial gastric glands showing atrophy or atypia. A large number of inflammatory cells were infiltrated the submucosa. Additionally, a significant number of curved or short rod-shaped, purple-blue clusters of Helicobacter pylori were visible. The serum levels of TNF-α and IL-6 were significantly increased (P<0.01). The protein expression levels of TLR5, TRAF6, TAK1, IKKα, NF-κB, MyD88, NF-κB p50 protein and NF-κB p65 protein were elevated (P<0.05). Furthermore, the gut microbiota dysbiosis was observed (P<0.05). Compared with those of the model group, the average body weight and damp-heat syndrome of the low, medium and high groups of Xianglian Pills were improved (P<0.05). The gastric mucosal epithelial cells were arranged in a relatively orderly way, with reduced glandular atrophy and atypia, as well as a significant decrease in inflammatory cell infiltration in the submucosa. The serum levels of TNF-α and IL-6 were significantly down-regulated (P<0.01). The protein expression levels of TLR5, TRAF6, TAK1, IKKα, NF-κB, MyD88, NF-κB p50 protein and NF-κB p65 protein were reduced (P<0.05). Additionally, the abundance of beneficial gut bacteria increased (P<0.05).

-Xianglian Pills can significantly improve gastric mucosal injury in HPAG rats with damp-heat in spleen-stomach syndrome, and its specific mechanism may be related to the regulation of TLR5/NF-κB signaling pathway and gut microbiota.