The therapeutic effect of Qishen Huoxue Granule on myocardial injury in sepsis rats and its underlying mechanism via suppressing excessive autophagy

Categories: Disease & Virus

September 2, 2025

Differential gene expression and network pharmacology analysis

A total of 1188 differentially expressed mRNAs were identified between disease-contrast normal specimens, thereinto, with 621 mRNAs upregulated and 567 mRNAs downregulated in disease. A volcano plot was drawn using the R package (ggplot2, version 3.3.5) to show the differential expression of the genes (shown in Fig. 1A). The R package (pheatmap, version 1.0.12) package was used to map the expression of differential genes in normal samples and disease samples (Fig. 1B). We first found 189 ingredients from TCMSP database, 125 from Angelica, 202 from Salvia miltiorrhiza, 119 from red peony, 189 from safflower and 87 from Astragalus. Meanwhile, 231 active components and 289 targets were selected according to OB 30% and DL 0.18. In the GeneCards database, 1551 targets were predicted to obtain 353 septic cardiomyopathy targets according to “Relevance score ≥ 5”.

The intersection of drug targets, disease targets, differential genes, and the results are shown in the Venn diagram, 18 key target genes were obtained: CHRM2, EDN1, SERPINE1, PPARA, ICAM1, TP53, INSR, STAT3, EDNRA, MYC, VCAM1, CXCL8, CAV1, GJA1, SELE, CCL2, SPP1, PYGM (no interaction with other proteins). Figure 1C, D.

Each of the 18 core target genes were co-enriched to 955 GO processes, among them, 930 are biological processes (BP), 5 are cellular components (CC), and 19 are molecular functions (MF). The main enrichment was for GO terms such as hypoxia response, response to reduced oxygen levels, and LPS response (Fig. 1E). KEGG was enriched to 108 pathways, mainly to: diabetic complications AGE-RAGE signaling pathway, lipid and atherosclerosis signaling pathway, PI3K-AKT-mTOR signaling pathway and other KEGG pathways (Fig. 1F).

QHG prolonged the survival in sepsis rats

The composition of traditional Chinese medicine includes Astragali Radix, Salvia Miltiorrhizae Radix, Angelica sinensis Radix, Paeoniae Rubra Radix, Chuanxiong Rhizoma, and Carthami Flos (Fig. 2A). A detailed flowchart of the in vivo assays was shown in Fig. 2B. The survival time of each group was recorded and the survival rate was calculated until 24 h after molding (Fig. 2C). Survival analysis showed that the 24 h survival rate of the CLP group of rats was 30%. Compared with the rats in the CLP group, the 24 h survival rate was increased to 80% in the CLP + QHG group. Compared with the CLP + QHG group, the 24 h survival rate decreased to 60% in the CLP + QHG + A779 group; The 24 h survival rate declined to nearly 60% in the CLP + QHG + Wortmannin group, while approaching 65% in the CLP + QHG + Rapamycin group.

QHG can improve myocardial function in sepsis

Body surface ultrasound detected the LVIDs, LVIDd, LVEDs, LVEDd, LVAWs, LVAWd, LVPWs, and LVPWd. LVEF and LVFS were calculated to evaluate left ventricular contraction in septic rats (Fig. 3A). Representative echocardiogram is shown in Fig. 3B, and LVEF and LVFS are shown in Fig. 3C. LVEF and LVFS were normal in the Sham group. Compared with the Sham group, the CLP group had a compensatory enhanced cardiac systolic function upon stimulation of sepsis, showing a significant increase in LVEF and LVFS Fig. 3B,C (P < 0.05). LVEF and LVFS were significantly lower in the CLP + QHG group compared with the CLP group (P < 0.05). However, pharmacological inhibition of these pathways using A779, Wortmannin, and Rapamycin abolished QHG’s beneficial effects, reaffirming the pathway’s role in QHG-mediated myocardial protection.

QHG ameliorates the myocardial pathological changes in sepsis

Under the light microscope, it can be seen that myocardial tissue structure of the Sham group rats is intact, with orderly arrangement of myocardial cells, no obvious swelling, deformation or necrosis, and no rupture of myocardial fibers. In the CLP group, myocardial cell rupture and necrosis increased, interstitial vascular dilation was significant, vascular wall thickening was observed, and myocardial fibers were broken. The CLP + QHG group showed mild swelling of myocardial cells, congestion and dilation of myocardial interstitium, and no significant myocardial cell shrinkage or nuclear membrane rupture. The myocardial fibers in the CLP + QHG + A779 group, CLP + QHG + Wortmannin group and CLP + QHG + Rapamycin group were unevenly arranged, with nuclear pyknosis, deformation, or disappearance, and significant myocardial interstitial edema (Fig. 4A).

Transmission electron microscopy revealed distinct ultrastructural differences among the experimental groups. In the Sham group, cardiomyocytes exhibited well-organized architecture with intact cellular structures, characterized by closely packed and regularly arranged mitochondrial cristae without evidence of mitochondrial swelling. In contrast, the CLP group displayed marked morphological alterations, including irregular myocyte thickness, pronounced mitochondrial swelling, structural disruption, and disorganized or indistinct mitochondrial cristae. The CLP + QHG group demonstrated significant preservation of cellular architecture, with cardiomyocytes maintaining orderly arrangement and mitochondria showing relatively intact membranes and clear structural organization. However, in the CLP + QHG + A779, CLP + QHG + Wortmannin, and CLP + QHG + Rapamycin groups, partial disruption of cellular organization was observed, manifested as disordered myocyte arrangement, variable thickness, mitochondrial swelling, and fragmented or indistinct cristae (Fig. 4B).

QHG improves myocardial markers and serum inflammatory factors in sepsis

As shown in Fig. 5A, the cTnT and BNP were significantly increased in the CLP group as compared to the Sham group (P < 0.05). Compared with the CLP + QHG group, the cTnT and BNP of CLP + QHG + A779, CLP + QHG + Wortmannin and CLP + QHG + Rapamycin were significantly increased (P < 0.05).

As illustrated in Fig. 5B, the CLP group exhibited markedly elevated serum levels of TNF-α and IL-1β compared to the Sham group (P < 0.05). QHG treatment effectively attenuated this inflammatory response, as evidenced by significantly reduced TNF-α and IL-1β levels in the CLP + QHG group relative to the CLP group (P < 0.05). However, there was no significant difference in serum inflammatory factors TNF-α and IL-1β between CLP + QHG + A779 group compared with CLP + QHG group. However, the CLP + QHG + Wortmannin group exhibited slightly elevated serum levels of IL-1β, whereas TNF-α levels compared to the CLP + QHG group were not significantly different. Notably, Rapamycin treatment (CLP + QHG + Rapamycin group) significantly reversed the anti-inflammatory effects of QHG, leading to substantial increases in serum TNF-α and IL-1β levels compared to the CLP + QHG group (P < 0.05).

QHG inhibits excessive autophagy in septic myocardial tissue

Immunohistochemical analysis revealed significant alterations in autophagy-related protein expression (Fig. 6A). Compared with the Sham group, the CLP group exhibited a marked increase in LC3 protein expression (P < 0.05). QHG treatment effectively downregulated LC3 expression (P < 0.05), whereas pharmacological inhibition of the MasR/PI3K-AKT-mTOR pathway reversed this effect, leading to significant upregulation of LC3 expression (P < 0.05). This trend is also observed in immunofluorescence (Fig. 6D).

The expression levels of autophagy markers (ATG5, Beclin1, and LC3) were quantitatively assessed at both mRNA and protein levels using RT-qPCR and Western Blot analyses, respectively (Fig. 6B and C). The uncut original image of Western Blot experiment is included in Supplementary Images S1 and S2.

While the Sham group maintained moderate autophagy activity, the CLP group exhibited significant upregulation of autophagy-related markers, with ATG5, Beclin1, and LC3 mRNA levels showing substantial elevation (P < 0.05). QHG treatment effectively attenuated this response, significantly reducing the expression of ATG5, Beclin1, and LC3 (P < 0.05). These findings demonstrate that QHG downregulates key autophagy indicators, providing compelling evidence for its role in suppressing excessive autophagy in septic rats. In addition, pharmacological inhibition of the MasR/PI3K-AKT-mTOR pathway reversed these effects, as evidenced by the increased mRNA and protein expression of ATG5 and Beclin1, indicating that the pathway inhibition counteracted QHG’s suppressive effect on autophagy.

QHG activates MasR/PI3K-AKT-mTOR pathway

We have performed molecular docking analyses to explore the binding affinity of key bioactive compounds in QHG (e.g., Tanshinone IIA, Geniposide, Hydroxysafflor yellow A) with core targets of the MasR/PI3K-AKT-mTOR pathway (MasR, PI3K, AKT, and mTOR). These results are now included as Supplementary Figure S3.

The expression profiles of key components in the MasR/PI3K-AKT-mTOR signaling pathway, including MasR, PI3K, AKT, and mTOR, were systematically evaluated at both mRNA and protein levels using RT-qPCR and Western Blot analyses (Fig. 7A-I). Quantitative analysis revealed significant downregulation of MasR, PI3K, AKT, and mTOR mRNA expression in the CLP group compared to Sham controls (P < 0.05), demonstrating substantial inhibition of this signaling pathway in septic myocardial tissue. QHG treatment effectively reversed this suppression, as evidenced by significantly elevated mRNA levels of MasR, PI3K, AKT, and mTOR in the CLP + QHG group relative to the CLP group (P < 0.05), indicating pathway activation.

However, pharmacological inhibition of the MasR/PI3K-AKT-mTOR pathway attenuated these effects, resulting in decreased mRNA expression of pathway components and reduced protein phosphorylation ratios (p-PI3K/PI3K, p-AKT/AKT, and p-mTOR/mTOR) (P < 0.05). These collective findings demonstrate that the cardioprotective effects of QHG against excessive autophagy are mediated through activation of the MasR/PI3K-AKT-mTOR signaling pathway, as pathway inhibition effectively counteracts these protective mechanisms.

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