SEQUENTIAL ANTIINFLAMMATORY EFFECTS OF LONGZUANTONGBI EXTRACT THROUGH REGULATION OF THE HMGB1–TLR4 SIGNALLING PATHWAY

Xiaoyue Chen*, Qinghuai Zhang**, Lan Huang***, Zongting Liang****, Jing Tang*, Zuling Jiang****, Zhiquan Wei*****, Yuzhou Pang******,#, Gang Fang*****, #

* International Hospital of Zhuang Medicine Affiliated, Guangxi University of Chinese Medicine, No.8, Qiu Yue Road, Nanning City, Guangxi, China, 530001 - **Zhuang Medicine College, Guanxi University of Chinese Medicine, No.179, Mingxiu East Road, Nanning City, Guangxi, China, 530001 - ***Graduate School, Guanxi University of Chinese Medicine, No.179, Mingxiu East Road, Nanning City, Guangxi, China, 530001 - ****The First Clinical Faculty, Guangxi University of Traditional Chinese Medicine, No.89-9, Dongge Road, Nanning City, Guangxi, China, 530023 - *****Laboratory of Zhuang Medicine Prescriptions Basis and Application Research, Guangxi University of Chinese Medicine, No.179, Mingxiu East Road, Nanning City, Guangxi, China, 530001 - ******Laboratory of Zhuang Medicine Prescriptions Basis and Application Research, Guangxi University of Chinese Medicine; Guangxi Zhuang Yao Medicine Center of Engineering and Technology, Guangxi University of Chinese Medicine, No.179, Mingxiu East Road, Nanning City, Guangxi, China, 530001

Introduction: The purpose of the present research was to study the anti-inflammatory effects of the Zhuang medicine Longzuantongbi decoction and its influence on the HMGB1–TLR4 signalling pathway. 

Materials and methods: Out of 120 SD rats, 30 were randomly selected as the normal group and injected with 0.2 mL of normal saline (NS) each time, whilst the remaining 90 SD rats were injected with Freund’s complete adjuvant once a week for 2 weeks. After 2 weeks, rats scoring over three points on the arthritis symptom scale were selected for successful modelling. Rats with successful modelling were randomly divided into a model group, low-dose group or high-dose group using different administration methods. Each group was further divided into 7-, 14- and 28-day (d) subgroups based on different administration times, with each group containing 10 rats. The normal and model groups were given 20 mL/kg of NS via intragastric administration. The low- and high-dose groups received 3 g/d and 6 g/d of Longzuantongbi decoction via intragastric administration, respectively. The rats were anesthetised at 7, 14 and 28 d after administration (once/day), and 5 mL of blood was collected so for the serum to be used for detection purposes. After the animals were sacrificed, the synovium was removed, soaked and fixed using methanol. The body posture changes and arthritis symptom scores of rats in each group were also recorded. Serum IL-1β and TNF-α levels were detected using the ELISA method, whilst synovium HMGB1, TLR4 and NF-κB protein levels were detected using western blotting. 

Results: Rats in the normal group were healthy and showed no obvious inflammatory lesions in the joints and bones. In the model group, acute inflammation initially occurred in the feet and ankles and then gradually developed into a systemic inflammatory response. The main manifestations were redness and swelling of the limbs, ears and tail as well as nodular inflammation. Moreover, arthritis index scores were also higher in these animals. Rats in the low- and high-dose groups showed symptoms that were prolonged with increasing administration time; however, the symptoms gradually improved, and the scoring significantly decreased (P<0.05), with improvement being most obvious in the high-dose group. IL-1β and TNF-α expression levels were low in the normal group, whilst HMGB1, TLR4 and NF-κB proteins were barely expressed. Inflammatory factors such as IL-1β, TNF-α, HMGB1, TLR4 and NF-κB proteins were expressed more in the model group than in the normal group (P<0.05). After administration, IL-1β, TNF-α, HMGB1, TLR4 and NF-κB expression levels significantly decreased in the low- and high-dose groups (P<0.05), with the decrease in the high-dose group being significantly greater than that of the low-dose group (P<0.05). The longer the administration time, the greater the dose and the lower the expression levels of various indices (P<0.05). 

Conclusion: The Longzuantongbi decoction can inhibit the release of inflammatory factors (i.e. IL-1β and TNF-α) and inhibit cellular signalling pathways (i.e. HMGB1–TLR4–NF-κB expression) to control the occurrence and development of rheumatoid arthritis and relieve its symptoms. The longer the medication time, the more significant the efficacy.