Shufeiya Recipe Improves Monocrotaline-Induced Pulmonary Hypertension in Rats by Regulating SIRT3/FOXO3a and Its Downstream Signaling Pathways

Abstract

Pulmonary hypertension (PH) is a chronic and progressive disease caused by obstructions and functional changes of small pulmonary arteries. Current treatment options of PH are costly with patients needing long-term taking medicine. The traditional Chinese medicine (TCM) compound "Shufeiya Recipe" was used to intervene in monocrotaline- (MCT-) induced pulmonary hypertension in rats. The rats were randomly divided into the control group, model group, positive drug (Sildenafil) group, and Shufeiya Recipe low-, moderate-, and high-dose groups. The improvement effect of the Shufeiya Recipe on the mean pulmonary artery pressure (mPAP) was assessed in PH rats, and pathological staining was used to observe the pathological changes of lung tissue. The impact of the Shufeiya Recipe on oxidative stress damage in rats with pulmonary hypertension and the regulation of SIRT3/FOXO3a and its downstream signaling pathways were determined. The results showed that Shufeiya Recipe could significantly downregulate mPAP and improve lung histopathological changes; downregulate serum levels of reactive oxygen species (ROS); upregulate the concentrations of COX-1 and COX-2 and the activity of Mn-SOD; inhibit oxidative response damage; promote the protein expression of SIRT3, FOXO3a, p-PI3K, p-AKT, and p-eNOS; increase the level of expression of NO, sGC, cGMP, and PKG; and downregulate the level of protein expression of Ras, p-MEK1/2, p-ERK1/2 and c-fos. These results indicate that Shufeiya Recipe can improve MCT-induced pulmonary hypertension in rats by regulating SIRT3/FOXO3a and its downstream PI3K/AKT/eNOS and Ras/ERK signaling pathways.

Figure 1

Shufeiya Recipe significantly relieved mPAP and pathological changes of lung tissue of PH rats. (a) Moderate and high doses of Shufeiya Recipe significantly decreased mPAP of PH rats. (b) Shufeiya Recipe relieved pathological changes of lung tissue of PH rats. n = 10; ^##P < 0.01 vs. the control group, ^∗∗P < 0.01 vs. the model group, and ^△△P < 0.01 vs. the moderate-dose group of Shufeiya Recipe.

Figure 2

Shufeiya Recipe significantly inhibited the oxidative stress level of PH rats. (a) Shufeiya Recipe significantly downregulated the level of ROS accumulation of PH rats, n = 3. (b–d) Shufeiya Recipe increased COX-1, COX-2, and Mn-SOD contents of PH rats, n = 6. ^##P < 0.01 vs. the control group; ^∗∗P < 0.01 vs. the model group; and ^△P < 0.05, ^△△P < 0.01 vs. the low-dose group of Shufeiya Recipe.

Figure 3

Shufeiya Recipe activated the SIRT3/FOXO3a signaling pathway of PH rats. (a) The high-dose group of Shufeiya Recipe stimulated the expression of FOXO3a. (b) The moderate- and high-dose groups of Shufeiya Recipe upregulated the expression level of SIRT3, and the most marked improvement was obtained by the high-dose treatment of Shufeiya Recipe. n = 3; ^##P < 0.01 vs. the control group; ^∗P < 0.05, ^∗∗P < 0.01 vs. the model group; and ^△P < 0.05 vs. the low-dose group of Shufeiya Recipe.

Figure 4

Shufeiya Recipe activated the PI3K/AKT/eNOS signaling pathway of PH rats. (a, b) Moderate and high groups of Shufeiya Recipe upregulated the relative immunofluorescence intensity of p-PI3K and p-eNOS. (c–e) Different doses of Shufeiya Recipe upregulated the protein expression level of p-PI3K, p-AKT, and p-eNOS. n = 3, ^##P < 0.01 vs. the control group; ^∗P < 0.05, ^∗∗P < 0.01 vs. the model group; ^△P < 0.05, ^△△P < 0.01 vs. the low-dose group of Shufeiya Recipe; ^※P < 0.05 vs. the moderate-dose group of Shufeiya Recipe.

Figure 5

Shufeiya Recipe upregulated concentration of NO, sGC, and cGMP and the protein expression level of PKG in PH rats. (a) Moderate and high groups of Shufeiya Recipe upregulated the protein expression level of PKG, n = 3. (b) Moderate and high groups of Shufeiya Recipe upregulated the concentration of NO, n = 3. (c, d) Moderate and high groups of Shufeiya Recipe upregulated the concentration of sGC and cGMP, n = 6. ^##P < 0.01 vs. the control group; ^∗P < 0.05, ^∗∗P < 0.01 vs. the model group; ^△P < 0.05 vs. the low-dose group of Shufeiya Recipe; and ^※P < 0.05 vs. the moderate-dose group of Shufeiya Recipe.

Figure 6

Shufeiya Recipe inhibited the Ras/ERK signaling pathway of PH rats. (a, b) Moderate and high groups of Shufeiya Recipe downregulated the relative immunofluorescence intensity of p-ERK1/2. (c–e) Different doses of Shufeiya Recipe downregulated the protein expression level of Ras, p-MEK1/2, p-ERK1/2, and c-fos. n = 3; ^##P < 0.01 vs. the control group; ^∗P < 0.05, ^∗∗P < 0.01 vs. the model group; ^△P < 0.05 vs. the moderate-dose group of Shufeiya Recipe.

Figure 7

The protective mechanism of Shufeiya Recipe regulated PH