[Pathogenesis of chronic heart failure in rats based on ferroptosis-mediated oxidative stress and intervention effect of Shenfu Injection].

This study aims to investigate the pathogenesis of chronic heart failure based on ferroptosis-mediated oxidative stress and predict the targets of Shenfu Injection in treating chronic heart failure. A rat model of chronic heart failure was established by the isoproterenol induction method. According to the random number table method, the modeled rats were assigned into three groups: a model group, a Shenfu Injection group, and a ferrostatin-1(ferroptosis inhibitor) group. In addition, a normal group was designed. After 15 days of intervention, the cardiac mass index and left ventricular mass index were determined. Echocardiography was employed to eva-luate the cardiac function. Hematoxylin-eosin staining and Masson staining were employed to reveal the pathological changes and fibrosis of the heart, and Prussian blue staining to detect the aggregation of iron ions in the myocardial tissue. Transmission electron microscopy was employed to observe the mitochondrion ultrastructure in the myocardial tissue. Colorimetry was adopted to measure the levels of iron metabolism, lipid peroxidation, and antioxidant indicators. Flow cytometry was employed to measure the content of lipid-reactive oxygen species(ROS) and the fluorescence intensity of ROS. Western blot and RT-qPCR were employed to determine the protein and mRNA levels, respectively, of ferroptosis-related factors in the myocardial tissue. The results showed that the rats in the model group had reduced cardiac function, elevated levels of total iron and Fe~(2+), lowered level of glutathione(GSH), increased malondialdehyde(MDA), decreased superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px), and rising levels of ROS and lipid-ROS. In addition, the model group showed fibrous tissue hyperplasia with inflammatory cell infiltration and myocardial fibrosis, iron ion aggregation, and characteristic mitochondrial changes specific for iron death. Moreover, the model group showcased upregulated protein and mRNA levels of p53 and COX2 and downregulated protein and mRNA levels of GPX4, FTH1, SLC7A11, and Nrf2 in the myocardial tissue. The intervention with Shenfu Injection significantly improved the cardiac function, recovered the iron metabolism, lipid peroxidation, and antioxidant indicators, decreased iron deposition, improved mitochondrial structure and function, and alleviated inflammatory cell infiltration and fibrosis. Furthermore, Shenfu Injection downregulated the mRNA and protein levels of p53 and COX2 and upregulated the mRNA and protein levels of GPX4, FTH1, SLC7A11, and Nrf2 in the myocardial tissue. Shenfu Injection can improve the cardiac function by regulating iron metabolism, inhibiting ferroptosis, and reducing oxidative stress injury.

[Protective effect of Shenfu Injection on regulation of autophagy in rats with chronic heart failure based on PI3K/Akt/mTOR pathway].

This study aimed to investigate the mechanism and target sites of Shenfu Injection in the intervention of chronic heart fai-lure based on the PI3K/Akt/mTOR autophagy signaling pathway. The chronic heart failure model was induced in rats by subcutaneous injection of isoproterenol. The model rats were randomly divided into model group, Shenfu Injection group, and 3-methyladenine autophagy inhibitor(3-MA) group. A normal group was also set up. After 15 days of administration, cardiac function indexes of the rats were detected by echocardiography. The serum N-terminal pro-B-type natriuretic peptide(NT-proBNP) levels were measured using the ELISA. HE and Masson staining was performed to observe the morphological changes in myocardial tissues, and electron microscopy was used to observe the autophagosomes in myocardial tissues. Western blot was conducted to measure the changes in autophagy-related proteins(LC3 Ⅱ/Ⅰ and p62), PI3K, Akt, mTOR, and phosphorylation levels. The results showed that compared with normal group, model group in rats led to reduced cardiac function, significant activation of cardiac autophagy, increased fibrotic lesions in myocardial tissues, structural disorder of the myocardium, increased autophagosomes, and cytoplasmic vacuolization. Compared with model group, Shenfu Injection group in rats led to cardiac function significantly improved, myocardial fibrosis decreased, and the number of autophagosomes and cytoplasmic vacuolization decreased. The phosphorylation levels of PI3K, Akt, and mTOR were significantly increased(P<0.01). In the 3-MA group, autophagy was inhibited through the activation of the PI3K/Akt/mTOR signaling pathway, resulting in improved cardiac function, reduced myocardial fibrosis, and no significant cytoplasmic vacuolization. The findings suggest that Shenfu Injection can activate the PI3K/Akt/mTOR signaling pathway and inhibit autophagy, thereby improving cardiac function.

Shenmai Injection Improves Hypertensive Heart Failure by Inhibiting Myocardial Fibrosis via TGF-ß 1/Smad Pathway Regulation.

OBJECTIVE: To study effects of Shenmai Injection on hypertensive heart failure and its mechanism for inhibiting myocardial fibrosis. METHODS: Salt-sensitive (Dahl/SS) rats were fed with normal diet (0.3% NaCl) and the high-salt diet (8% NaCl) to observe the changes in blood pressure and heart function, as the control group and the model group. Salt-insensitive rats (SS-13BN) were fed with the high-salt diet (8% NaCl) as the negative control group. After modeling, the model rats were randomly divided into heart failure (HF) group, Shenmai Injection (SMI) group and pirfenidone (PFD) group by a random number table, with 6 rats in each group. They were given sterilized water, SMI and pirfenidone, respectively. Blood pressure, cardiac function, fibrosis and related molecular expression were detected by sphygmomanometer, echocardiogram, enzyme linked immunosorbent assay (ELISA), hematoxylin-eosin staining, Masson staining, immunofluorescence and qPCR analysis. RESULTS: After high-salt feeding, compared with the control and negative control group, in the model group the blood pressure increased significantly, the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were significantly reduced, and the serum NT-proBNP concentration increased significantly (all P<0.05); furthermore, the arrangement of myocardial cells was disordered, the edema was severe, and the degree of myocardial fibrosis was also significantly increased (P<0.05); the protein and mRNA expressions of collagen type I (Col I) were up-regulated (P<0.05), and the mRNA expressions of transforming growth factor ß 1 (TGF- ß 1), Smad2 and Smad3 were significantly up-regulated (P<0.05). Compared with HF group, after intervention of Shenmai Injection, LVEF and LVFS increased, myocardial morphology was improved, collagen volume fraction decreased significantly (P<0.05), and the mRNA expressions of Col I, TGF- ß 1, Smad2 and Smad3, as well as Col I protein expression, were all significantly down-regulated (all P<0.05). CONCLUSION: Myocardial fibrosis is the main pathological manifestation of hypertensive heart failure, and Shenmai Injection could inhibit myocardial fibrosis and effectively improve heart failure by regulating TGF-ß 1/Smad signaling pathway.

[Shenfu Injection improves chronic heart failure by regulating pyroptosis based on NLRP3/caspase-1 pathway].

This study investigated the mechanisms and targets of Shenfu Injection in the intervention in chronic heart failure(CHF) through the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/caspase-1 signaling pathway. A CHF model was induced in rats by subcutaneous injection of isoproterenol. Model rats were randomly divided into a model group, a Shenfu Injection group, and a MCC950(NLRP3 inhibitor) group, and a blank group was also set up as a control. After 15 days of treatment, echocardiography was performed to measure cardiac function parameters [left ventricular ejection fraction(LVEF) and left ventricular fractional shortening(LVFS)]. Enzyme-linked immunosorbent assay(ELISA) was used to measure serum levels of N-terminal pro-brain natriuretic peptide(NT-proBNP), interleukin(IL)-1ß, and IL-18. Hematoxylin-eosin(HE) and Masson staining were used to observe morphological changes in myocardial tissues, and Western blot was used to measure the expression levels of NLRP3/caspase-1 pathway-related proteins [NLRP3, caspase-1, apoptosis-associated speck-like protein containing a CARD(ASC), gasdermin D(GSDMD), IL-1ß, and IL-18]. The study found that isoproterenol-induced CHF in rats resulted in decreased cardiac function, worsened myocardial fibrosis, increased expression levels of NLRP3, ASC, caspase-1, GSDMD-N, IL-1ß, and IL-18 in myocardial tissues, elevated serum inflammatory factors, and induced myocardial cell pyroptosis. Following Shenfu Injection intervention, the Shenfu Injection group showed significantly improved LVEF and LVFS, a significant decrease in NT-proBNP, a marked downregulation of NLRP3, ASC, caspase-1, GSDMD-N, IL-1ß, and IL-18 protein expression levels, reduced serum inflammatory factors IL-1ß and IL-18 expression in CHF rats, and a decrease in the rate of TUNEL-positive cells. Shenfu Injection can significantly improve cardiac function in CHF, inhibit myocardial fibrosis, and alleviate the progression of myocardial cell pyroptosis through the inhibition of the NLRP3/caspase-1 pathway.

[Protective effect of Shenfu Injection on rats with chronic heart failure based on HMGB1/TLR4/NF-κB signaling pathway].

The study aimed to explore the mechanism and targets of Shenfu Injection in the regulation of inflammatory injury in chronic heart failure rats based on the high mobility group box-1/Toll like receptor 4/nuclear factor kappa-B(HMGB1/TLR4/NF-κB) signaling pathway. The rat model of chronic heart failure was established using isoproterenol. The modeled rats were divided into three groups by random number table: the model group, Shenfu group and glycopyrrolate group, and the normal group was also set. The rats were administrated for 15 consecutive days, and on the following day after the last administration, they were sacrificed for sample collection. The cardiac mass index and left ventricular mass index of the rats in each group were measured, and the echocardiogram was used to analyze the cardiac function indices, and ELISA to test the inflammatory indices in rat serum. The pathological morphology and fibrosis status of rat heart tissues were observed by HE staining and Masson staining, respectively. The content of HMGB1 was determined by immunofluorescence staining. The protein and mRNA expression of HMGB1/TLR4/TLR4 signaling pathway was detected by Western blot and RT-qPCR, respectively. The results showed that the chronic heart failure rat model was successfully prepared. The rats in the model group had reduced cardiac function, increased levels of HMGB1 and inflammatory factors(P<0.05), and elevated protein and mRNA expression of HMGB1, TLR4, MyD88, and NF-κB P65 in myocardial tissue(P<0.05), with fibrous connective tissue hyperplasia, inflammatory cell infiltration and severe fibrosis. Shenfu Injection improved cardiac function, decreased the levels of HMGB1 and inflammatory factors(P<0.05) and the protein and mRNA expression of HMGB1, TLR4, MyD88, and NF-κB P65 in myocardial tissue(P<0.05), ameliorated interstitial fibrous connective tissue hyperplasia and inflammatory cell infiltration, and reduced fibrosis. In conclusion, Shenfu Injection can reduce inflammatory damage and improve cardiac function in chronic heart failure rats by regulating the HMGB1/TLR4/NF-κB signaling pathway.

[Animal model analysis of rheumatoid arthritis based on clinical characteristics of Chinese and Western medicine].

Rheumatoid arthritis(RA) is an autoimmune disease involving multiple joints bilaterally with symmetrical polyarthritis as the main symptom. The high disability rate of this disease seriously affects the quality of life of patients and even threatens their lives. The establishment of a good animal model is of great significance for the diagnosis and clinical prevention of RA. Based on the clinical characteristics of RA in traditional Chinese and Western medicine, the common animal models of RA were summarized, including drug-induced, gene-related, and syndrome and disease combined models. Joint swelling, pain, redness, nodules, and joint deformity are the main criteria for model evaluation, which have certain differences from the clinical diagnostic criteria of RA. From the perspective of syndrome differentiation, the animal model combining syndrome and disease only simulates the syndrome of traditional Chinese medicine and has no direct causal relationship with the formation of RA. In this paper, we analyzed the advantages and disadvantages of animal models of RA and the coincidence degree of the models with the clinical characteristics and then put forward the corresponding recommendations for the evaluation and improvement of these models, aiming to make the animal models of RA closer to the clinical symptoms and play an important role in the clinical diagnosis and treatment of RA.

[Analysis of animal models of hypertension based on characteristics of clinical symptoms of Chinese and western medicine].

Hypertension, a cardiovascular disease with main clinical manifestations of dizziness and elevated blood pressure, especially elevated arterial pressure, features high prevalence rate and low control rate, which affects patients' quality of life. Therefore, establishing a good animal model of hypertension is of great significance for its diagnosis and clinical prevention and treatment. Based on the clinical characteristics of hypertension in traditional Chinese and western medicine, this study summarized the advantages and disadvantages of current hypertension animal models: gene-related model, surgery-caused model, drug-induced model, and environment-induced model, and investigated the similarity to the clinical symptoms in traditional Chinese medicine and Western medicine. Among them, spontaneously hypertensive rats, models established with the surgical two-kidney one-clip, one-kidney one-clip, two-kidney two-clip, and abdominal aorta constriction methods, models induced with the drug deoxycorticosterone acetate, and models induced with the high-fat high-purine diet showed symptoms highly similar to the clinical manifestations. Then, the corresponding evaluation and improvement methods of hypertension animal models were proposed. This study provides suggestions for the establishment of hypertension animal model so that the symptoms are more similar to the clinical characteristics of hypertension in traditional Chinese and Western medicine, which is important for the clinical diagnosis and treatment of hypertension.