Effect of a combination of Atractylodes macrocephala extract with strychnine on the TLR4/NF-κB/NLRP3 pathway in MH7A cells

Abstract

Rheumatoid arthritis (RA) is now widely recognized as a chronic systemic inflammatory autoimmune disease characterized by swelling, pain and stiffness, which are often disabling. Although the number of drugs available for the treatment of RA has increased in recent years, they are generally expensive, leave patients prone to relapse and can result in severe effects when discontinued. Thus, there is a need for an inexpensive drug with fewer side effects that can be adhered to relieve pain and slow down the progression of the disease. Strychnine, a traditional Chinese medicine, was often used in ancient times to treat swollen and painful joints; however, because of its somewhat toxic nature, it is often combined with Atractylodes macrocephala to reduce its toxicity for safer therapeutic action. The present study performed high-performance liquid chromatography (HPLC)-tandem mass spectrometry (MS/MS) analysis to confirm whether the use of strychnine with Atractylodes macrocephala had the effect of reducing strychnine content. MH7A cells were induced using IL-1β to study the effect of strychnine with Atractylodes macrocephala on the Toll-like receptor 4 (TLR4)/NF-κB/NLR family pyrin domain-containing 3 (NLRP3) pathway in order to verify its role in the treatment of RA. The results indicated that the combined application of HPLC-MS/MS strychnine and Atractylodes macrocephala had a reducing effect on the strychnine content. From the subsequent experimental results, it can be inferred that Strychnine combined with Atractylodes macrocephala extract could promote the apoptosis of synovial cells, and could inhibit the expression levels of TLR4, NF-κB and NLRP3 in the cells as well as reducing the MH7A-positive cells. The expression levels of TLR4, IκB kinase β, NF-κB and NLRP3 were significantly reduced after treatment with each administration group, resulting in a decrease in the phosphorylation levels of TLR4 and NF-κB, indicating that the combination potently inhibited their phosphorylation. The combination of strychnine and atractylenolide II was also revealed to be the main active ingredient in the treatment of RA.

Keywords: Atractylodes macrocephala; MH7A cells; NF-κB; NLR family pyrin domain-containing 3; Toll-like receptor 4; brucine; rheumatoid arthritis; strychnine.

Figure 1

Image and chemical structure of strychnine and Atractylodes macrocephala alongside HPLC-MS/MS chromatography. (A) Strychnine chemical structure and formula of Strychnine and Brucine. (B) Atractylodes macrocephala chemical structure and formula of Atractylodes II. (C) HPLC-MS/MS chromatography (mixed standard, strychnine soup, strychnine and Atractylodes macrocephala 1:6 and strychnine and Atractylodes macrocephala 1:12). HPLC-MS/MS, high performance liquid chromatography with tandem mass spectrometry; AM group, the mixed standard. SS group, the liquid obtained by decoction of strychnine (20 g); SA (1:6) group, liquid from decoction of strychnine (20 g) and Atractylodes macrocephala (120 g); SA (1:12), liquid from decoction of strychnine (20 g) and Atractylodes macrocephala (240 g).

Figure 2

Cell Counting Kit-8 assay. Effect of different concentrations of strychnine and Atractylenolide II on the proliferation of MH7A cells at the same time. ^*P<0.05 and ^**P<0.01 vs. Control.

Figure 3

Images of the cell morphology after 24 h. MTX, Strychnine, Atractylodes macrocephala; S:A 1:6, co-decoction of two drugs, Strychnine and Atractylodes macrocephala; S:A 1:6 component group, extract of strychnine and Atractylenoloid II.

Figure 4

Apoptosis of MH7A cells following treatment with strychnine combined with Atractylodes macrocephala. n=5. ^#P<0.05 and ^##P<0.01 vs. Model. MTX, Strychnine, Atractylodes macrocephala; S:A 1:6, co-decoction of two drugs, Strychnine and Atractylodes macrocephala; S:A 1:6 component group, extract of strychnine and Atractylenoloid II.

Figure 5

Immunofluorescence analysis of TLR4, NF-κB and NLRP3 expression and localization in MH7A cells following treatment with the strychnine and Atractylodes macrocephala combination. n=3. ^**P<0.01 vs. Control; ^#P<0.05 and ^##P<0.01 vs. induced by IL-1β. TLR4, Toll-like receptor 4; NF-κB; NLRP3, NLR family pyrin domain-containing 3 expression; MTX, Strychnine, Atractylodes macrocephala; S:A 1:6, co-decoction of two drugs, Strychnine and Atractylodes macrocephala; S:A 1:6 component group, extract of strychnine and Atractylenoloid II.

Figure 6

Effect of strychnine with Atractylodes macrocephala on IL-1β-mediated mRNA expression of TLR4, IKKβ, NF-κBp65, NLRP3 and TNF-α in MH7A cells. n=5. ^**P<0.01 vs. Control; ^##P<0.01 vs. Induced by IL-1β. TLR4, Toll-like receptor 4; IKK, IκB kinase; NF-κBp65; NLRP3, NLR family pyrin domain-containing 3; TNF-α expression; MTX, Strychnine, Atractylodes macrocephala; S:A 1:6, co-decoction of two drugs, Strychnine and Atractylodes macrocephala; S:A 1:6 component group, extract of strychnine and Atractylenoloid II.

Figure 7

Effect of the strychnine and Atractylodes macrocephala combination on IL-1β-mediated expression of TLR4, NF-κB, IKKβ, NF-κB p65, p-NF-κB p65, NLRP3, p-NLRP3 and TNF-α in MH7A cells. n=5. ^**P<0.01 vs. Control; ^#P<0.05 and ^##P<0.01 vs. Induced by IL-1β. TLR4, Toll-like receptor 4; IKK, IκB kinase; p-NF-κB p65; NF-κB p65. NLRP3, NLR family pyrin domain-containing 3; p-, phosphorylated; TNF-α expression;, MTX, methotrexate; Strychnine; Atractylodes macrocephala; S: A 1:6 co-decocted of two drugs co-decocted of two drugs, Strychnine, and Atractylodes macrocephala, S: A 1:6 extract of Strychnine and Atractylodes macrocephala is strychnine and Atractylenoloid II component group.

Figure 8

Diagram of the TLR4/NF-κB/NLRP3 signaling pathway. IKK, IκB kinase; NLRP3, NLR family pyrin domain-containing 3; TLR4, Toll-like receptor 4; PYD, pyrin domain; LRR, leucine-rich repeat domain; ASC, apoptosis-associated speck-like protein; CARD, caspase recruitment domain; FC11A-2, 1-ethyl-5-methyl-2-phenyl-1H-benzo[d]imidazole.