Xinfeng Capsule Inhibits Pyroptosis and Ameliorates Myocardial Injury in Rats with Adjuvant Arthritis via the GAS5/miR-21/TLR4 Axis

Figures & data

Table 1 Primer Used for RT-qPCR

Targets	Sequences
LncRNA GAS5	Forward:5’-AAGCTCCACACAAGGTCCTT-3’
	Reverse: 5’-GCATCCATCCAGTCACCTCT-3’
NLRP3	Forward: 5’-GAAACTCTGGTTGGTCAGC-3’
	Reverse: 5’-AGAGAATGGGTTGGAGCTCAG-3’
GSDMD	Forward: 5’-CGACTCTGGAGAACTGGTG-3’
	Reverse: 5’-TGGGTTTCACTCAACCCAG-3’
Caspase-1	Forward: 5’-AAGATGATGGCATTAAGAAGGC-3’
	Reverse: 5’-TCCAGGACACATTATCTGGTG-3’
TLR4	Forward: 5’-CCTGCATAGAGGTACTTCCT-3’
	Reverse: 5’-AATAAGGGGATGTCATGAGGGA-3’
NF-κB p65	Forward: 5’-TGTGAAGAAGCGAGACCTG-3’
	Reverse: 5’-TCCTCTATGGGAACTTGAAAGG-3’
GAPDH	Forward: 5’-AGGTCGGGTGTGAACGGATTTG-3’
	Reverse: 5’-GGGGTCGTTGATGGCAACA-3’
miR-21	Forward: 5’-GCGCGTAGCTTATCAGACTGA-3’
	Reverse: 5’-AGTGCAGGGTCCGAGGTATT-3’
miR-21 (reverse transcription)	5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACA-3’
U6	Forward: 5’-CTCGCTTCGGCAGCACA-3’
	Reverse: 5’-AACGCTTCACGAATTTGCGT-3’

Figure 1 Effect of XFC on toe swelling and arthritis index in AA rats.

Notes: (A) Toe swelling degree of rats in each group. (B) Arthritis index of rats in each group. NC, the normal control group; MC, the model control group; XFC, the Xinfeng Capsule group; MTX, the methotrexate tablet group; XFC + EV, the Xinfeng Capsule + empty AAV group; XFC + GAS5, the Xinfeng Capsule + AAV harboring GAS5 overexpression group; XFC + NSA, the Xinfeng Capsule + NSA (a pyroptosis inhibitor) group. n=number of rats (6 rats/group).

Figure 2 Effects of XFC on the histopathology of synovium and myocardium in AA rats (HE, ×200).

Notes: (A) Synovial histopathology of rats. (B) Myocardial histopathology of rats. NC, the normal control group; MC, the model control group; XFC, the Xinfeng Capsule group; MTX, the methotrexate tablet group; XFC + EV, the Xinfeng Capsule + empty AAV group; XFC + GAS5, the Xinfeng Capsule + AAV harboring GAS5 overexpression group; XFC + NSA, the Xinfeng Capsule + NSA (a pyroptosis inhibitor) group.

Figure 3 Effects of XFC on the ultrastructure of myocardial in AA rats (TEM, ×6000).

Notes: The ultrastructure of the myocardium in different groups of rats is shown in the figure. The red arrow refers to the pyroptotic vesicles. NC, the normal control group; MC, the model control group; XFC, the Xinfeng Capsule group; MTX, the methotrexate tablet group; XFC + EV, the Xinfeng Capsule + empty AAV group; XFC + GAS5, the Xinfeng Capsule + AAV harboring GAS5 overexpression group; XFC + NSA, the Xinfeng Capsule + NSA (a pyroptosis inhibitor) group.

Figure 4 Effect of XFC on serum cytokines in AA rats.

Notes: (A) IL-1β levels in different groups. (B) IL-18 levels in different groups. (C) IL-6 levels in different groups. (D) TNF-α levels in different groups. NC, the normal control group; MC, the model control group; XFC, the Xinfeng Capsule group; MTX, the methotrexate tablet group; XFC + EV, the Xinfeng Capsule + empty AAV group; XFC + GAS5, the Xinfeng Capsule + AAV harboring GAS5 overexpression group; XFC + NSA, the Xinfeng Capsule + NSA (a pyroptosis inhibitor) group. Values are mean ± standard deviation, n=number of rats (6 rats/group). **P < 0.01, compared with the NC group; ^##P < 0.01, ^#P < 0.05, compared with the MC group; ^ΔΔP < 0.01, ^ΔP < 0.05, compared with the XFC group.

Figure 5 Effect of XFC on LDH release from myocardial tissues in AA rats.

Notes: The graph displays the statistical analysis results of LDH release from rat myocardial tissues in different groups. NC, the normal control group; MC, the model control group; XFC, the Xinfeng Capsule group; MTX, the methotrexate tablet group; XFC + EV, the Xinfeng Capsule + empty AAV group; XFC + GAS5, the Xinfeng Capsule + AAV harboring GAS5 overexpression group; XFC + NSA, the Xinfeng Capsule + NSA (a pyroptosis inhibitor) group. Values are mean ± standard deviation, n=number of rats (6 rats/group). **P < 0.01, compared with the NC group; ^##P < 0.01, ^#P < 0.05, compared with the MC group; ^ΔΔP < 0.01, compared with the XFC group.

Figure 6 Effects of XFC on the expression of the GAS5/miR-21/TLR4 axis and pyroptosis-related indicators in myocardial tissues of AA rats.

Notes: (A) Protein levels of TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD in rat myocardial tissues. (B) Semi-quantitative analysis of protein levels. (C) Expression of GAS5 and mRNA expression of miR-21, TLR4, NF-κB p65, NLRP3, Caspase-1, and GSDMD in rat myocardial tissues. NC, the normal control group; MC, the model control group; XFC, the Xinfeng Capsule group; MTX, the methotrexate tablet group; XFC + EV, the Xinfeng Capsule + empty AAV group; XFC + GAS5, the Xinfeng Capsule + AAV harboring GAS5 overexpression group; XFC + NSA, the Xinfeng Capsule + NSA (a pyroptosis inhibitor) group. Values are mean ± standard deviation, n=number of rats (6 rats/group). **P < 0.01, compared with the NC group; ^##P < 0.01, ^#P < 0.05, compared with the MC group; ^ΔΔP < 0.01, compared with the XFC group.