Method for verifying protection effect of semen descurainiae aqueous extract on myocardial injury

Abstract

The invention provides a method for verifying the protective effect of a semen descurainiae water extract on myocardial injury, so as to evaluate the pharmacodynamic effect of the semen descurainiae water extract on heart failure induced by three inducements of acute ischemia-reperfusion injury, pathological cell hypertrophy and chronic hypoxia. The method comprises the following steps: respectively constructing a hiPSC-CMs-based myocardial acute ischemia reperfusion injury model, a pathological fat myocardial injury model and a chronic hypoxic myocardial injury model, and after the myocardial injury models are established, adding a descurainia seed aqueous extract and a positive control drug for intervention. And detecting corresponding indexes to verify the effect of the descurainia seed water extract on three types of heart failure. The result shows that the semen descurainiae water extract can generate a treatment effect on myocardial injury caused by chronic hypoxia by regulating and controlling the steady state of energy metabolism, inhibiting oxidative stress cascade reaction and other mechanisms.

Description

Technical Field

[0001] The present invention relates to the technical field of traditional Chinese medicine efficacy evaluation, and in particular to a method for verifying the protective effect of an aqueous extract of southern scutellaria baicalensis seeds on myocardial injury. Background Art

[0002] Descurainiae Semen (DS) is the dried mature seed of the cruciferous plant Artemisia selengensis, a traditional Chinese medicinal herb. Its pungent and bitter properties enter the lung, bladder, and large intestine meridians. It is primarily distributed in Northeast my country, as well as in Hebei, Shandong, Henan, and Zhejiang provinces. It contains chemical components such as glucosinolates, flavonoids, and cardiac glycosides, and has the effects of purging the lungs, relieving asthma, and promoting diuresis and reducing swelling.

[0003] In the cardiovascular field, there are only a few studies on Atractylodes macrocephala. For example, some studies have shown that the water extract of Atractylodes macrocephala has a significant protective effect on hydrogen peroxide (H2O2)-induced H9C2 rat embryonic cardiomyocyte damage. Its mechanism may be related to improving intracellular redox homeostasis and inhibiting mitochondrial apoptosis pathways and autophagy pathways.

[0004] Sun Zhiqiang et al. ［1］ SD rats were treated with abdominal aortic constriction surgery to establish a heart failure model. They were then given either a water or alcohol extract of Scutellaria baicalensis seeds as a preventive measure. The results showed that, compared with the model group, preventive administration improved left ventricular ejection fraction and fractional shortening, indicating that Scutellaria baicalensis seeds have a significant preventive effect on decreased myocardial contractile function caused by ischemia and hypoxia, but had no effect on diastolic blood pressure.

[0005] Fan Chunlan et al. ［2］ Using the same surgical procedure, Wistar rats were modeled for 8 weeks and then given medication for 4 weeks. The authors found that the aqueous / ethanol extract of (Southern) Tripterygium wilfordii seeds was effective in regulating both systolic and diastolic blood pressure and inhibiting overactivation of the neuroendocrine system in myocardial tissue. However, the study report did not provide any experimental data. These results are not entirely consistent, possibly due to species differences or differences in sample extraction methods.

[0006] However, the above studies cannot predict the clinical efficacy of the water extract of Atractylodes macrocephala. On the one hand, this is because there are huge differences between animal cardiomyocytes and human hearts in ion channels and characteristic proteins. On the other hand, the causes of clinical heart failure are often multi-event and multi-factorial, which require more comprehensive consideration.

[0007] References: [1] Sun Zhiqiang, Li Chao, Wu Yuanhong, et al. Effects of extract of southern scutellaria baicalensis seeds on cardiac function in rats with heart failure [J]. Journal of Prevention and Treatment of Cardiovascular and Cerebrovascular Diseases, 2016, 16(06): 427-430.

[0008] [2] Fan Chunlan, Yu Yingmei, Zhang Yanzhuo. Effects of extract of scutellaria baicalensis seeds on hemodynamics and plasma AngⅡ and ALD levels in rats with CHF[J]. Chinese Journal of Ethnomedicine, 2009, 18(22): 8-9. Summary of the Invention

[0009] The purpose of the present invention is to provide a method for verifying the protective effect of the water extract of southern scutellaria baicalensis on myocardial injury, so as to evaluate the pharmacological effect of the water extract of southern scutellaria baicalensis on heart failure induced by three causes: acute ischemia-reperfusion injury, pathological cell hypertrophy, and chronic hypoxia.

[0010] The technical solution is as follows: A method for verifying the protective effect of an aqueous extract of scutellaria baicalensis seeds on myocardial injury comprises the following steps: (1) Using hiPSC-CMs and establishing a myocardial injury model after spontaneous contraction occurs, wherein the myocardial injury model is selected from: a. Acute myocardial ischemia-reperfusion injury model: hiPSC-CMs were placed in a serum-free, sugar-free medium at 0.49% oxygen for 2 hours, then returned to normoxia and replaced with serum-containing cardiomyocyte medium for 24 hours. b. Hypertrophic myocardial injury model: hiPSC-CMs were induced to hypertrophy with 0.1 μM Ang II for 72 hours; c. Chronic hypoxic myocardial injury model: hiPSC-CMs were cultured in an oxygen concentration of 11.98% for 144 hours. (2) After the myocardial injury model was established, the aqueous extract of the fruit of the Chinese angelica was added to the culture medium at a concentration gradient of 1 to 100 μg / mL, and the positive control drug was 100 μM trimetazidine or captopril; (3) Evaluate the efficacy of the aqueous extract of the seeds of the Chinese angelicae dahuricae by testing at least one of the following indicators: i. Cell apoptosis rate; ii. Mitochondrial functional integrity; iii. Oxidative stress levels; iv. Spontaneous contraction function; v.Cell area; vi. Hypertrophy gene expression; (4) Verify the intervention effect of the water extract of southern scutellaria baicalensis on myocardial injury based on the changes in corresponding indicators.

[0011] Preferably, based on the acute myocardial ischemia-reperfusion injury model: drug intervention in the reperfusion phase is carried out simultaneously when the cells recover to normoxic conditions, and the exposure time of the water extract of southern scutellaria baicalensis fruit or trimetazidine is 24 hours.

[0012] Preferably, based on the hypertrophic myocardial injury model: drug intervention is performed 72 hours after Ang II induction, and the exposure time of the water extract of the seeds of the southern scutellaria or captopril is 24 hours.

[0013] Preferably, based on the chronic hypoxic myocardial injury model: drug intervention is performed after 96 hours of exposure to a hypoxic environment, the exposure time of the water extract of the southern scutellaria baicalensis fruit or trimetazidine is 48 hours, and the hypoxic environment will continue until the end of the experiment.

[0014] Preferably, the detection of cell apoptosis rate includes: Annexin V-FITC / PI staining was used to quantify the proportion of apoptotic cells.

[0015] Preferably, the detection of mitochondrial functional integrity comprises: Mitochondrial morphology is marked by the Mito-Tracker fluorescent probe.

[0016] Preferably, the detection of the oxidative stress level comprises: DCFH-DA probe detects reactive oxygen species accumulation; The mRNA expression levels of antioxidant and pro-oxidant genes were determined by qRT-PCR.

[0017] Preferably, the detection of spontaneous contraction function includes: A high-speed camera system records the contraction rate and rhythmicity; The mRNA expression levels of contraction-specific genes were determined by qRT-PCR.

[0018] Preferably, the detection of the cell area comprises: Phalloidin staining was used to detect cell area; The detection of hypertrophy gene expression includes qPCR analysis of hypertrophy marker gene expression.

[0019] Preferably, the 100 μg / mL concentration group of the water extract of the southern scutellaria baicalensis fruit has no significant difference from the positive control drug in the following indicators: The cell survival rate recovered to more than 50% of that in the normoxia group; ROS levels recovered to within 1.5 times that of the normoxic group; The contraction rate recovered to more than 65% of the normoxia group.

[0020] Compared with the existing technology, the beneficial effects of the present invention are: utilizing hiPSC-CMs and constructing a myocardial injury model after spontaneous contraction occurs: first, a myocardial acute ischemia-reperfusion injury model is constructed, and the effect of the water extract of Achyranthes bidentata seeds on this type of heart failure is verified by detecting four indicators, namely, cell apoptosis rate, mitochondrial function, oxidative stress level and spontaneous contraction function; second, a pathological hypertrophic myocardial injury model is constructed, and the effect of the water extract of Achyranthes bidentata seeds on this type of heart failure is verified by detecting three aspects, namely, hypertrophy markers, oxidative stress level and spontaneous contraction function of cells; third, a chronic hypoxic myocardial injury model is constructed, and the effect of the water extract of Achyranthes bidentata seeds on this type of heart failure is verified by detecting four indicators, namely, cell apoptosis rate, mitochondrial function, oxidative stress level and spontaneous contraction function; the three models can more comprehensively reflect the three causes of heart failure.

[0021] Results showed that treatment with the A. scutellariae fruit water extract ameliorated multidimensional damage in the model group. Apoptosis decreased, mitochondrial network structural integrity and energy metabolism activity were restored, the accumulation of oxidative stress markers decreased, and regular spontaneous beating resumed. These results suggest that the A. scutellariae fruit water extract may have a therapeutic effect on chronic hypoxia-induced myocardial injury by regulating energy metabolism homeostasis and inhibiting the oxidative stress cascade. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The effect of DS on myocardial cell apoptosis induced by ischemia-reperfusion injury is shown in Example 1; Figure 2 The effect of DS on mitochondria of myocardial cells injured by ischemia-reperfusion is shown in Example 1; Figure 3 The effect of DS on ROS in myocardial cells injured by ischemia-reperfusion is shown in Example 1; Figure 4 The effect of DS on the spontaneous contraction of myocardial cells injured by ischemia-reperfusion is shown in Example 1; Figure 5 The effect of DS on the area of ​​hypertrophic heart failure cells in Example 2 is shown; Figure 6 The second embodiment shows the effect of DS on the marker genes of hypertrophic heart failure cells; Figure 7 The second embodiment shows the effect of DS on ROS in hypertrophic heart failure cells; Figure 8 The second embodiment shows the effect of DS on the spontaneous contraction of hypertrophic heart failure cells; Figure 9 The third embodiment shows the effect of DS on the apoptosis of myocardial cells under chronic hypoxia. Figure 10 The third embodiment shows the effect of DS on mitochondria of chronic hypoxic cardiomyocytes; Figure 11 The third embodiment shows the effect of DS on ROS in chronic hypoxic cardiomyocytes; Figure 12 The third embodiment shows the effect of DS on the spontaneous contraction of chronic hypoxic cardiomyocytes. DETAILED DESCRIPTION

[0023] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0024] Example 1: Effect of the aqueous extract of Tengli Fructus on acute myocardial ischemia-reperfusion injury 1. Construction of acute myocardial ischemia-reperfusion injury model Cell source: Human induced pluripotent stem cell-differentiated cardiomyocytes (hiPSC-CMs), which were used in experiments after their spontaneous contractile function was confirmed.

[0025] Ischemia model: cells were transferred to a hypoxic environment (0.49% O2) and replaced with serum-free and sugar-free medium for 2 h.

[0026] Reperfusion treatment: restore normoxia (21% O2) and regular serum-containing culture medium to simulate clinical reperfusion conditions.

[0027] 2. Experimental Grouping The cells were divided into 5 groups, with 3 biological replicates in each group: ①Normoxia group: cultured in 21% O2+ conventional culture medium throughout the whole process.

[0028] ② Ischemia group: hypoxia (0.49% O2) + serum-free medium treatment for 2 h.

[0029] ③ Ischemia-reperfusion group (I / R group): After 2 hours of ischemia, the rats were restored to standard conditions for 24 hours.

[0030] ④ DS intervention group: 1, 10, and 100 μg / mL of scutellaria baicalensis fruit aqueous extract (DS) were added during reperfusion.

[0031] ⑤Positive control group: 100 μM trimetazidine (TMZ) was added during reperfusion.

[0032] 3. Detection indicators Cell apoptosis: Annexin V-FITC / PI staining was used to quantify the proportion of apoptotic cells.

[0033] Mitochondrial morphology and function: Mitochondrial distribution and fluorescence intensity were observed by Mito-Tracker Red staining.

[0034] Oxidative stress: ROS levels were detected using a DCFH-DA probe; the expression of antioxidant (NQO1, SOD2) and pro-oxidant genes (NOX2, NOX4, NOXA2) was analyzed by qRT-PCR.

[0035] Contractile function: Spontaneous contraction rate was recorded by video analysis system; the expression levels of contraction-specific gene mRNA (RYR2, ATP2A2) were measured by qRT-PCR.

[0036] 4. Test results ①DS inhibits ischemia-reperfusion-induced apoptosis, see Figure 1 shown.

[0037] The survival rate in the I / R group dropped significantly from 90% in the normoxia group to 39%.

[0038] The survival rate of the DS intervention group increased with increasing concentration (48%, 56%, 57%), and there was no statistically significant difference between the 100 μg / mL DS group and the TMZ group (64%).

[0039] ②DS improves mitochondrial structure and function, see Figure 2 shown.

[0040] The mitochondrial fluorescence intensity in the I / R group decreased by 62% compared with the normoxia group, and showed perinuclear aggregation; the fluorescence intensity in the DS-treated group recovered by 5-10%, which was consistent with the trend in the TMZ group.

[0041] ③DS regulates the balance of oxidative stress, see Figure 3 shown.

[0042] The ROS level in the I / R group increased to 2.34 compared with the normoxia group (0.99), was inhibited in a dose-dependent manner in the DS group (2.22, 1.87, 1.53), and was further reduced to 1.29 in the TMZ group.

[0043] DS significantly upregulated the expression of antioxidant factors (NQO1, SOD2) and downregulated the expression of pro-oxidative factors (NOX2, NOX4, NOXA2). The effects of the 100 μg / mL DS group were comparable to those of the TMZ group, with no significant difference, confirming that DS can alleviate damage by regulating the oxidative stress pathway.

[0044] ④DS restores myocardial cell contractile function, see Figure 4 shown.

[0045] The contraction rate in the I / R group decreased by 51% compared with the normoxia group. The contraction rate reduction in the 1, 10, and 100 μg / mL DS treatment groups improved to 39%, 37%, and 34%, respectively, and reached 30% in the TMZ group.

[0046] Genetic testing showed that DS alleviated the inhibition of RYR2 and ATP2A2 expression (increased by 10-20% compared with the I / R group), and its dose-dependent recovery trend was consistent with the functional data, suggesting that DS improves contractile dysfunction by maintaining calcium-regulating gene expression.

[0047] 5. Conclusion The aqueous extract of Scutellaria baicalensis seeds (DS) alleviates ischemia-reperfusion injury in hiPSC-CMs through the following mechanisms: ① Anti-apoptotic effect: DS increased cell survival rate in a dose-dependent manner, and the effect of 100 μg / mL DS was comparable to that of TMZ.

[0048] ② Mitochondrial protection: maintain the integrity of the mitochondrial network and energy metabolism activity.

[0049] ③Anti-oxidative stress: regulate the expression of redox-related genes and reduce ROS accumulation.

[0050] ④ Functional recovery: Improve calcium regulatory gene expression and partially reverse contractile dysfunction.

[0051] After DS treatment, the multidimensional damage of the model group cells was improved: the apoptosis rate was reduced, the structural integrity of the mitochondrial network and energy metabolism activity were restored, the accumulation of oxidative stress markers was reduced, and regular spontaneous beating was restored. These results suggest that southern sedge seeds may have a therapeutic effect on myocardial ischemia-reperfusion injury by regulating energy metabolism homeostasis and inhibiting the oxidative stress cascade.

[0052] Example 2: Effect of the Aqueous Extract of Southern Tetrapanax chinensis (DS) on Hypertrophic Myocardial Injury 1. Construction of hypertrophic myocardial injury model Cell source: Human induced pluripotent stem cell-differentiated cardiomyocytes (hiPSC-CMs), which were used in experiments after their spontaneous contractile function was confirmed.

[0053] Hypertrophy model: The cell culture medium was replaced with cardiomyocyte culture medium containing 0.1 μM Ang II and the treatment was continued for 72 h to induce cardiomyocyte hypertrophy.

[0054] 2. Experimental Grouping The cells were divided into 5 groups, with 3 biological replicates in each group: ①Control group: Cultured with conventional culture medium throughout the whole process.

[0055] ②Hypertrophy model group: Ang II (0.1 μM) treated for 72 h.

[0056] ③DS intervention group: After Ang II-induced hypertrophy, the mice were treated with 1, 10, and 100 μg / mL DS for 24 h.

[0057] ④ Positive control group: After Ang II-induced hypertrophy, the cells were treated with 100 μM captopril (Cap) for 24 h.

[0058] 3. Detection indicators Cell area: Phalloidin staining was used to detect the cell area.

[0059] Cell hypertrophy: qPCR analysis of the expression of hypertrophy marker genes (ANP, BNP, β-MHC).

[0060] Oxidative stress: ROS levels were detected using a DCFH-DA probe; the expression of antioxidant (NQO1, SOD2) and pro-oxidant genes (NOX2, NOX4, NOXA2) was analyzed by qRT-PCR.

[0061] Contractile function: Spontaneous contraction rate was recorded by video analysis system; the expression levels of contraction-specific gene mRNA (RYR2, ATP2A2) were measured by qRT-PCR.

[0062] 4. Test results ①DS inhibits Ang II-induced cardiomyocyte hypertrophy, see Figure 5 and Figure 6 shown.

[0063] The cell area in the Ang II model group increased significantly by 1.7 times compared with the control group.

[0064] The cell area in the DS treatment groups (1, 10, and 100 μg / mL) was reduced by 0.14, 0.27, and 0.33 times, respectively, compared with the model group. There was no statistically significant difference between the 100 μg / mL DS group and the Cap group (reduced by 0.47 times).

[0065] DS inhibited the expression of hypertrophic genes (ANP, BNP, β-MHC) in a dose-dependent manner, which was consistent with the regulatory trend of the Cap group.

[0066] ②DS reduces oxidative stress damage, see Figure 7 shown.

[0067] The ROS fluorescence intensity in the Ang II model group increased to 1.7 compared with that in the control group (1.0).

[0068] The ROS levels in the DS treatment groups (1, 10, and 100 μg / mL) were reduced to 1.62, 1.51, and 1.44, respectively. The ROS levels in the 100 μg / mL DS group were comparable to those in the Cap group (1.31).

[0069] The DS treatment group significantly upregulated antioxidant factors (NQO1, SOD2) and downregulated pro-oxidative factors (NOX2, NOX4, NOXA2). For example, in the 100 μg / mL DS treatment group, NQO1 and SOD2 increased to 0.57 and 0.74, respectively, while NOX2, NOX4, and NOXA2 decreased to 3.32, 4.46, and 2.51, respectively, which was consistent with the trend of the Cap group.

[0070] ③DS restores myocardial cell contractile function, see Figure 8 shown.

[0071] After 72 hours of Ang II treatment, the cardiomyocytes in each group showed significant inhibition of contractile function, and the contraction rate was lower than that of the control group; if Ang II treatment was continued for another 24 hours, the contraction rate of the cardiomyocytes would further decrease, which was 9% lower than that after 72 hours of Ang II treatment.

[0072] After 24 h of DS treatment, the contraction rate increased by 63% (1 μg / mL), 93% (10 μg / mL), and 108% (100 μg / mL) compared with the model group, respectively. There was no significant difference between the 100 μg / mL DS group and the Cap group (113%).

[0073] DS restored the expression of RYR2 and ATP2A2 in a dose-dependent manner (increased by 10-20% compared with the model group), and its trend was consistent with the functional data.

[0074] 5. Conclusion The aqueous extract of Scutellaria baicalensis (DS) alleviates Ang II-induced hypertrophic myocardial injury in hiPSC-CMs through the following mechanisms: Anti-hypertrophic effect: significantly reduced cell area and hypertrophic marker gene expression, and the effect of 100 μg / mL DS was comparable to that of Cap.

[0075] Anti-oxidative stress: regulate redox balance, reduce ROS levels and the expression of pro-oxidative factors.

[0076] Functional recovery: Improves calcium regulatory gene expression and reverses contractile dysfunction.

[0077] After DS treatment, the multidimensional damage of the model group cells was improved: cell area decreased, expression of hypertrophy marker genes decreased, accumulation of oxidative stress markers decreased, and regular spontaneous beating was restored. These results demonstrate that southern sedge seed has a therapeutic effect on Ang II-induced cardiomyocyte hypertrophy.

[0078] Example 3: Effects of water extract of scutellaria baicalensis seeds (DS) on chronic hypoxic myocardial injury 1. Construction of a chronic hypoxic myocardial injury model Cell source: Human induced pluripotent stem cell-differentiated cardiomyocytes (hiPSC-CMs), which were used in experiments after their spontaneous contractile function was confirmed.

[0079] Chronic hypoxia model: The cells were placed in a hypoxic environment (11.98% O2) and cultured continuously for 144 h to establish a chronic hypoxic myocardial injury model through continuous hypoxia exposure.

[0080] Drug intervention: After 96 h of hypoxia exposure, 1, 10, and 100 μg / mL DS and 100 μM TMZ were added, respectively, and hypoxic conditions (11.98% O2) were maintained for another 48 h.

[0081] 2. Experimental Grouping The cells were divided into 5 groups, with 3 biological replicates in each group: ①Normoxia control group: Cultured under standard culture conditions (21% O2 + conventional culture medium) throughout the whole process.

[0082] ②Hypoxia model group: 11.98% O2 treatment for 144 h.

[0083] ③ DS intervention group: After 96 h of hypoxia, 1, 10, and 100 μg / mL DS were added for 48 h.

[0084] ④ Positive control group: After 96 h of hypoxia, 100 μM trimetazidine (TMZ) was added for 48 h.

[0085] 3. Detection indicators Cell apoptosis: Annexin V-FITC / PI staining was used to quantify the proportion of apoptotic cells.

[0086] Mitochondrial function: Mitochondrial distribution and fluorescence intensity were observed by Mito-Tracker Red staining.

[0087] Oxidative stress: ROS levels were detected using a DCFH-DA probe; the expression of antioxidant (NQO1, SOD2) and pro-oxidant genes (NOX2, NOX4, NOXA2) was analyzed by qRT-PCR.

[0088] Contractile function: Spontaneous contraction rate was recorded by video analysis system; the expression levels of contraction-specific gene mRNA (RYR2, ATP2A2) were measured by qRT-PCR.

[0089] 4. Test results ①DS inhibits chronic hypoxia-induced apoptosis, see Figure 9 shown.

[0090] The survival rate of the normoxic control group was 87%, and its background apoptosis rate may be attributed to the inherent physiological consumption of the in vitro culture system. The survival rate of the hypoxic model group dropped significantly to 71%, a significant decrease of 16% compared with the control group, confirming that hypoxic stress can induce pathological cell death processes.

[0091] The survival rate of the DS intervention group (1, 10, 100 μg / mL) was 5-10% higher than that of the model group (77%, 78%, 81%), and that of the TMZ group was 82%.

[0092] There was no statistical difference between the 100 μg / mL DS group and the TMZ group, showing a similar improvement trend.

[0093] ②DS restores mitochondrial structure and function, see Figure 10 shown.

[0094] The mitochondrial fluorescence intensity in the hypoxia model group decreased by 70% compared with the normoxia group, and the distribution was perinuclear aggregation.

[0095] The fluorescence intensity of the DS-treated group recovered by 10-50% (10%, 30%, 50%), and that of the TMZ group recovered by 54%, suggesting that DS can repair mitochondrial membrane integrity and functional activity, and the TMZ group showed similar mitochondrial protective effects.

[0096] ③DS regulates the balance of oxidative stress, see Figure 11 shown.

[0097] The relative fluorescence intensity of ROS in the hypoxia model group was significantly increased by 1.3 times compared with that in the normoxia group.

[0098] The ROS levels in the DS-treated group dropped to 1.23 (1 μg / mL), 1.21 (10 μg / mL), and 1.08 (100 μg / mL), respectively, and that in the TMZ group was 1.13. After intervention with DS and the positive control drug TMZ, the ROS fluorescence intensity decreased to varying degrees, suggesting that the drugs have the biological effect of scavenging oxygen free radicals.

[0099] Chronic hypoxia caused a decrease in the relative mRNA expression levels of key antioxidant factors NQO1 and SOD2; at the same time, the expression levels of pro-oxidant factors NOX2, NOX4, and NOXA2 increased. DS significantly upregulated NQO1 (+35% compared with the model group) and SOD2 (+156%), and downregulated NOX2 (-11%), NOX4 (-28%), and NOXA2 (-21%). The TMZ group showed a similar regulatory trend. These results indicate that DS can restore the redox homeostasis of myocardial cells by enhancing the antioxidant defense system and inhibiting the production of reactive oxygen species driven by the NOX enzyme system.

[0100] ④DS improves myocardial cell contractile function, see Figure 12 shown.

[0101] The contraction rate of the hypoxia model group decreased by 25% compared with the normoxia group.

[0102] After DS intervention, the contraction rate increased by 12% (1 μg / mL), 33% (10 μg / mL), and 28% (100 μg / mL), respectively, while the TMZ group increased by 46%.

[0103] Further monitoring of contraction-related genes revealed that chronic hypoxia caused the relative mRNA expression levels of myocardial contraction-related genes RYR2 and ATP2A2 to decrease to 0.37 times and 0.26 times that of the normoxia group, respectively; DS dose-dependently restored the expression of RYR2 (+89% compared with the model group) and ATP2A2 (+23%), which was consistent with the improvement trend of the TMZ group.

[0104] 5. Conclusion The aqueous extract of southern scutellaria seeds (DS) alleviates chronic hypoxic myocardial injury through the following mechanisms: Anti-apoptotic effect: significantly improved cell survival rate, and the effect of 100 μg / mL DS was comparable to that of TMZ.

[0105] Mitochondrial protection: restore mitochondrial distribution and energy metabolism function.

[0106] Anti-oxidative stress: Restore redox homeostasis and inhibit ROS accumulation.

[0107] Functional recovery: Upregulation of calcium regulatory gene expression partially reverses contractile dysfunction.

[0108] The results indicate that southern sedge seeds may have a therapeutic effect on myocardial damage caused by chronic hypoxia by regulating energy metabolism homeostasis, inhibiting oxidative stress cascade reactions and other mechanisms.

[0109] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements can be made without departing from the inventive concept of the present invention, which all fall within the scope of protection of the present invention.

Claims

1. A method for verifying the protective effect of the aqueous extract of Acanthocephalae chinensis on myocardial injury, characterized in that: The following steps are involved: (1) Using hiPSC-CMs and establishing a myocardial injury model after spontaneous contraction occurs, wherein the myocardial injury model is selected from: a. Acute myocardial ischemia-reperfusion injury model: hiPSC-CMs were placed in a serum-free, sugar-free medium at 0.49% oxygen for 2 hours, then returned to normoxia and replaced with serum-containing cardiomyocyte medium for 24 hours. b. Hypertrophic myocardial injury model: hiPSC-CMs were induced to hypertrophy with 0.1 μM Ang II for 72 hours; c. Chronic hypoxic myocardial injury model: hiPSC-CMs were cultured in an oxygen concentration of 11.98% for 144 hours. (2) After the myocardial injury model was established, the aqueous extract of the fruit of the southern sedge was added to the culture medium at a concentration gradient of 1 to 100 μg / mL. The positive control drug was 100 μM trimetazidine or captopril. (3) Evaluate the efficacy of the aqueous extract of the seeds of the Chinese angelicae dahuricae by testing at least one of the following indicators: i. Cell apoptosis rate; ii. Mitochondrial functional integrity; iii. Oxidative stress levels; iv. Spontaneous contraction function; v.Cell area; vi. Hypertrophy gene expression; (4) Verify the intervention effect of the water extract of southern scutellaria baicalensis on myocardial injury based on the changes in corresponding indicators.

2. The method for verifying the protective effect of the aqueous extract of Acanthocephalae chinensis on myocardial injury according to claim 1, characterized in that: Based on the acute myocardial ischemia-reperfusion injury model: drug intervention in the reperfusion phase was carried out simultaneously when the cells recovered to normoxic conditions, and the exposure time of the water extract of southern scutellaria or trimetazidine was 24 hours.

3. The method for verifying the protective effect of the aqueous extract of Acanthocephalae chinensis on myocardial injury according to claim 1, characterized in that: Based on the hypertrophic myocardial injury model: drug intervention was carried out 72 hours after Ang II induction, and the exposure time of the water extract of southern scutellaria or captopril was 24 hours.

4. The method for verifying the protective effect of the aqueous extract of Acanthocephalae chinensis on myocardial injury according to claim 1, characterized in that: Based on the chronic hypoxic myocardial injury model: drug intervention is carried out after 96 hours of exposure to a hypoxic environment, the exposure time of the water extract of the southern scutellaria or trimetazidine is 48 hours, and the hypoxic environment will continue until the end of the experiment.

5. The method for verifying the protective effect of the aqueous extract of Acanthocephalae chinensis on myocardial injury according to claim 1, characterized in that: The detection of the cell apoptosis rate includes: Annexin V-FITC / PI staining was used to quantify the proportion of apoptotic cells.

6. The method for verifying the protective effect of the aqueous extract of Acanthocephalae chinensis on myocardial injury according to claim 1, characterized in that: The detection of mitochondrial functional integrity includes: Mitochondrial morphology is marked by the Mito-Tracker fluorescent probe.

7. The method for verifying the protective effect of the aqueous extract of Acanthocereus chinensis on myocardial injury according to claim 1, characterized in that: The detection of the oxidative stress level includes: DCFH-DA probe detects ROS accumulation; The mRNA expression levels of antioxidant and pro-oxidant genes were determined by qRT-PCR.

8. The method for verifying the protective effect of the aqueous extract of Acanthocephalae chinensis against myocardial injury according to claim 1, characterized in that: The detection of the spontaneous contraction function includes: A high-speed camera system records the contraction rate and rhythmicity; The mRNA expression levels of contraction-specific genes were determined by qRT-PCR.

9. The method for verifying the protective effect of the aqueous extract of Acanthocereus chinensis on myocardial injury according to claim 1, characterized in that: The detection of the cell area comprises: Phalloidin staining was used to detect cell area; The detection of hypertrophy gene expression includes qPCR analysis of hypertrophy marker gene expression.

10. The method for verifying the protective effect of the aqueous extract of Acanthocereus chinensis on myocardial injury according to claim 1, characterized in that: The 100 μg / mL concentration group of the water extract of the southern scutellariae seeds showed no significant differences from the positive control drug in the following indicators: The cell survival rate recovered to more than 50% of that in the normoxia group; ROS levels recovered to within 1.5 times that of the normoxic group; The contraction rate recovered to more than 65% of the normoxia group.

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