Camel Whey protein ameliorates type Ⅰ diabetic cardiomyopathy by mitigating oxidative stress, inflammation and apoptosis

Abstract

The online version contains supplementary material available at 10.1007/s44463-025-00021-0.

Figures

Effect of CWP on myocardial hypertrophy in type Ⅰ diabetes mice. (A) Timeline for in vivo experiments. (B-C) WGA staining and quantitative analysis of myocardial tissue. (D-E) RNA expression levels of ANP and BNP. STZ, streptozotocin; ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide. *n* = 6 mice per group. * *p* < 0.05, *p* < 0.0001

Effect of CWP on cardiac inflammatory response in type Ⅰ diabetes mice. (A-F) RNA expression level of IL-6, TNF-α, MCP-1, ICAM-1, VCAM-1, IL-10. (G-H) IL-1β immunoblot analysis and quantification. *n* = 6 mice per group. * *p* < 0.05, *p* < 0.01, * *p* < 0.001, *p* < 0.0001

CWP inhibits oxidative stress in myocardial tissue of type I diabetes mice by activating Nrf2/HO-1 signaling pathway. (A-B) Activity of SOD and CAT in serum. (C-D) Activity of SOD and CAT in the heart tissues. (E-G) Western blotting and quantitative analysis of Nrf2, HO-1, SOD2 in the heart tissues. (H) RNA expression level of Nrf2 and HO-1 in the heart tissues. SOD, superoxide dismutase; CAT, catalase. *n* = 6 mice per group. * *p* < 0.05, *p* < 0.01, * *p* < 0.001, *p* < 0.0001

Effect of CWP on cardiomyocyte apoptosis. (A-B) Western blotting and quantitative analysis of Bax and Bcl-2. *n* = 4. * *p* < 0.05

Effect of CWP on NRVMS apoptosis and inflammatory response. (A-B) Detection of apoptotic cells by TUNEL staining, positive cells were indicated by a white arrow and quantification of apoptotic cells. (C-D) Western blotting and quantitative analysis of Bax and Bcl-2. (E) RNA expression level of IL-6, TNF-α, MCP-1, ICAM-1, VCAM-1, IL-10. NRVMs, neonatal rat ventricular myocytes; HG, 33 mM glucose. *n* = 4. * *p* < 0.05, *p* < 0.01, * *p* < 0.001, *p* < 0.0001

Effect of CWP on NRVMS myocardial hypertrophy and oxidative stress. (A) RNA expression levels of ANP and BNP. (B) Representative pictures of immunofluorescent staining for F-actin. (C) Quantification of cardiomyocyte size after CON、HG and CWP treatment. (D) RNA expression levels of Nrf2 and HO-1. (E) Superoxide dismutase was detected by DHE staining in NRVMs. (F-G) Western blotting and quantitative analysis of SOD2、Nrf2 and HO-1. *n* = 4. * *p* < 0.05, *p* < 0.01, * *p* < 0.001, *p* < 0.0001

Full text

Introduction

Diabetes cardiomyopathy (DCM), a typical complication of diabetes, is a major cause of diabetic demise accounting for more than 50% of diabetic deaths (Jia et al., 2018; Li et al., 2020). DCM is a specific cardiac disorder induced by insulin resistance, compensatory hyperinsulinemia, and progressive hyperglycemia (Ghosh et al., 2023). It is different from those of other common cardiovascular complications. DCM is characterized by myocardial dysfunction in diabetic patients without overt coronary artery disease, valvular disease, dyslipidemia, or systemic hypertension. In contrast, the core pathological process of coronary artery disease (CAD) involves lipid deposition, thrombus formation, and blood flow obstruction within the coronary arteries, leading to myocardial ischemia and ultimately resulting in angina or myocardial infarction (MI) (Zhang & Liu, 2024). MI, another common cardiovascular complication, is primarily caused by arterial thrombotic occlusion, characterized by a sudden reduction in blood flow to the myocardium, ultimately leading to heart failure and death. Its pathological mechanisms also differ significantly from those of DCM (Zhang et al., 2022).

Early intervention is crucial in the management of DCM. At present, some approaches involving multiple therapeutic strategies is applied combining multiple therapeutic strategies is required, including glycemic control, lipid regulation, and the use of β-adrenergic receptor antagonists (Adeghate et al., 2010). However, existing treatment modalities are associated with certain limitations and adverse effects. Metformin, a first-line antidiabetic agent, is known to cause gastrointestinal discomfort, lactic acidosis, and hepatotoxicity (Mian et al., 2023). Simvastatin, commonly used for the treatment of hyperlipidemia, can lead to a significant elevation in creatine kinase levels, resulting in muscle pain and an increased risk of hemorrhagic stroke (Collins et al., 2016). Additionally, β-adrenergic receptor blockers such as carvedilol have been reported to induce BRASH syndrome in some cases (Yan et al., 2024). Therefore, screening bioactive molecules with metabolic regulation potential and few side effects and clarifying their molecular mechanisms are of great significance for formulating prevention and treatment strategies for DCM (Li et al., 2020).

The pathophysiological mechanisms of DCM are complex, involving many aspects such as metabolic dysregulation and cardiac remodeling. Metabolic dysregulation in DCM is commonly characterized by hyperglycemia, insulin resistance, and abnormal fatty acid metabolism. Insulin resistance not only impairs glucose metabolism but also disrupts fatty acid metabolism in cardiomyocytes. Impaired insulin signaling reduces glucose utilization by cardiomyocytes, thereby increasing the metabolic burden on fatty acid oxidation. This shift leads to the accumulation of fatty acid metabolic byproducts in the myocardium, contributing to lipotoxicity and increased cardiac stress (Ghosh et al., 2023). In a hyperglycemic state, insulin resistance and dysregulated fatty acid metabolism can directly or indirectly exacerbate oxidative stress and inflammation, further leading to myocardial structural and functional abnormalities (Salvatore et al., 2021; Wilson et al., 2018). Chronic hyperglycemia promotes excessive accumulation of reactive oxygen species (ROS) in cardiomyocytes, establishing a state of oxidative stress. Additionally, it facilitates the accumulation of advanced glycation end products (AGEs) in diabetic patients. Oxidative stress and AGEs activate multiple inflammatory pathways, stimulating the release of pro-inflammatory cytokines such as transforming growth factor-β (TGF-β) and tumor necrosis factor-α (TNF-α). These factors promote the activation of cardiac fibroblasts, leading to enhanced collagen synthesis and deposition, ultimately resulting in myocardial fibrosis (Kaludercic & Di Lisa, 2020; Ni et al., 2016; Wilson et al., 2018). Oxidative stress and the strong inflammatory response triggered by oxidative stress play an important role in the occurrence and development of DCM. Therefore, agents which can alleviate oxidative stress and inflammation are assumed to be very prospective to retard the formation of DCM (Luo et al., 2020; Ren et al., 2020).

The nuclear factor erythroid2-related factor2 (Nrf2) is one of the therapeutic targets for DCM that can enhance the antioxidant, anti-inflammatory, anti-fibrosis, and anti-apoptosis effects in the myocardium. Its activation can increase the activities of related antioxidant enzymes and detoxification enzymes, such as superoxide dismutase, heme oxidase-1 (HO-1), and glutathione peroxidase. Earlier studies indicated that suppressing oxidative stress by activation of theNrf2/HO-1 pathway was beneficial for the survival of cardiomyocytes (Qin et al., 2019). The regulation of some vital pathways related to oxidative stress, such as Nrf2 signaling pathway, can effectively alleviate DCM (Li et al., 2019; Liu et al., 2017).

Camel milk (CM) is a major dairy product in droughty regions. It is considered as an optimal substitute for breast milk (Swelum et al., 2021). Camel whey protein (CWP) comprises 20–25% of the total camel milk proteins. It also contains a different group of proteins including serum albumin, α-lactoalbumin, immunoglobulin, lactoferrin, peptidoglycan recognition protein, lactoperoxidase, lysozyme and so on (Badr et al., 2017). In recent times, the attention of the scientific community has increasingly focused on CM and their bioactive compounds, largely because of their multiple health benefits. This comes against the backdrop of growing drug resistance and the significant side effects posed by synthetic medicines (Arain et al., 2023). There have been researches indicating that camel milk can alleviate type Ⅰ diabetes as it contains insulin and insulin-like substances as well as small-sized immunoglobulins (Zibaee et al., 2015). Furthermore, it has been found that CM lactoferrin and CM-derived bioactive peptides positively modulate insulin receptor and its related signaling pathways (Anwar et al., 2021). Lactoglobulin, lactoperoxidase and α-lactoalbumin have shown some medicinal properties, including antimicrobial, anti-inflammatory, antioxidant, anti-diabetic and immunomodulatory activities (Arain et al., 2023). CM also facilitate insulin level normalization, blood fats index regulation and antioxidant capacity enhancement in diabetic mice (Mohamad et al., 2009). Meanwhile, CWP has also been proved to be able to alleviate diabetes liver injury (Dou et al., 2023). However, the effect of camel milk or its component such as CWP in alleviating DCM is yet to be determined.

In this experiment, we used laboratory mice with type Ⅰ diabetes as an experimental model. CWP has been tested for the first time for its protective effect on the hearts of diabetic mice in terms of lipid metabolism, myocardial hypertrophy, inflammation, oxidative stress, signaling pathway and cardiomyocyte apoptosis.

Materials and methods

Preparation of CWP

Fresh camel milk provided by Xinjiang University was centrifugated at 8000 rpm for 10 min to eliminate fat, followed by filtration of impurities with clean 200-mesh gauze. The skimmed milk was then acidized to pH 4.6 at room temperature, heated in water bath at 40 ℃ for 20 min, cooled at 4 ℃for 4 h, finally centrifugated at 8000 rpm for 10 min in order to precipitate casein. The supernatant substance containing CWP was filtered, pH neutralized, freeze-dried and refrigerated for later use.

Animal experiments

All animal experiments were reviewed and approved by the Experimental Animal Management Committee of Nankai University (2023-SYDWLL-000389) and comply with the ARRIVE guidelines and are carried out in accordance with the U.K. Animals (Scientific Procedures) Act.

Eighteen 6-week-old male C57BL/6 mice weighing 20–30 g each were used for the experiment. They were placed in polypropylene cages with access to standard rodent diet and water under regulated circumstances (temperature 22 ± 2 °C, light/dark cycle 12 h). The main ingredients of the standard rodent diet are corn, soybean meal, calcium hydrogen phosphate, multiple vitamins, multiple trace elements, amino acids, etc. (3.02 kcal/gm). Then all the mice were acclimatized in the animal room for seven days before starting any experiment.

Diabetic mouse model preparation

In order to induce type Ⅰ diabetes, streptozotocin (STZ) (50 mg kg^− 1^ body weight) diluted in 0.1 mol l^− 1^ sodium citrate buffer (pH 4.5) was injected by intraperitoneal injection as described elsewhere (Zhang et al., 2020), one dose per day for 5 days. Meanwhile, the control group only received sodium citrate buffer by the same dose. Fasting glucose levels (FGLs) (4 h fasting) in experimental groups were observed on day 7 after STZ injection. The blood glucose level in the tail vein of mice detected by the blood glucose meter (Roche, Germany) was more than 13.8 mmol l^− 1^, enough to be diagnosed as diabetes. Thereby mouse models were successfully acquired and ready to be used in subsequent experiments.

Treatment on diabetic mice and organ preservation

The experimental subjects were divided into three groups of six mice in each group: control group (CON), diabetic group (DM) and diabetic group treated with CWP (CWP). The CWP group was fed by gavage with CWP (400 mg kg^− 1^ body weight) diluted in distilled water every day for six weeks. Both CON group and DM group were fed with the same quantity distilled water in the same way and at the same frequency. Feed and water were administered to each group of mice as described in the previous “Animal Experiments” section. The CWP dose was found to be 400 mg/kg after some preliminary experiments and also referred to previous research (Du et al., 2022).

The body weight and random blood glucose of mice in each group were measured and recorded once a week during the experiment. Twelve hours after the last gavage, mice were anesthetized with pentobarbital sodium salt (50 mg/kg). Blood samples were collected from the abdominal aorta in a non-heparinized tube, left at normal atmospheric temperature for two hours, centrifuged for 10 min at 3000 rpm to separate the serum, then stored at −80 °C and later used to biochemical analyses. The heads of surviving mice were then excised under anesthesia, the heart tissue was separated into two segments. The upper part namely the atrium was treated with 4% paraformaldehyde while the lower part containing the ventricles and part of the atrium was frozen at −80 °C for further research.

Biochemical measurements

Various components of the serum including cholesterol, triglyceride, low density lipoprotein (LDL), triglyceride (TG), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK) and creatine kinase isoenzyme (CKMB) were examined by the automatic biochemical analyzer (Abbott C16000; Sisen Meikang xn1000).

Superoxide dismutase and catalase assay

We measured superoxide dismutase (SOD) and catalase (CAT) levels of the heart and serum samples with a commercial kit (Nanjing Jiancheng Co., China). Take two 5 mg portions of heart tissue and immerse them in physiological saline at a ratio of 1:10. The tissue homogenate was processed with a high-throughput tissue grinder and after centrifugation, the supernatant substance was extracted for further examinations according to instructions.

Cell cultures

H9C2 cells were preserved by our lab. The cells were cultured in DMEM containing 10% fetal bovine serum, and cultured in an incubator containing 5% CO_2_ at 37 °C.

Extraction and cultivation of NRVMs

Neonatal rat ventricular myocytes (NRVMs) were obtained from rats born within 72 h (SPF biotechnology, Beijing, China). Hearts of neonatal rats were divided equally into three parts and placed in trypsin solution (1 mg/mL) for overnight digestion at 4 ℃. Afterwards, the liquid was pipetted and the digestion process was terminated using DMEM medium containing 7% FBS + 1% PS. Then the liquid was pipetted again and type Ⅱ collagenase (75 U/mL) was added. After digestion for 5–6 min, the tissue was blown with a pipette. Meanwhile the cell suspension was filtered and collected with a 70 μm nylon mesh and centrifugated at 500 rpm for 5 min. The cell mass was suspended on the medium and moved into a petri dish, where it was cultured twice in a humid environment at 37 °C with 5% CO_2_ for 75 min (fibroblasts were removed by differential adhesion). The supernatant layer was removed and distributed evenly in a petri dish, which remained in a carbon dioxide incubator for 24 h. Then the cells were cultured in a DMEM medium containing 1% PS + 1% ITS.

H9C2 cells and NRVMs were divided into three groups: control group (CON), high glucose group (HG) and high glucose treated with CWP (CWP). CWP group pre-treated with 1.5 mg/mL of CWP dissolved in DMEM (5.5 mM glucose); HG group and CON group processed with only DMEM (5.5 mM glucose) for 24 h. Then the culture medium of HG group was replaced by DMEM (33 mM glucose) while that of CWP group 1.5 mg/mL CWP dissolved in DMEM (33 mM glucose). All were cultured for 36 h before testing.

Western blotting

Ten milligram homogenized cardiac tissue and 3 × 10^6^ neonatal rat cardiomyocytes were dissolved with RIPA and centrifuged at 12,000 rpm for 30 min The supernatant was transferred to a new EP tube, a portion of the supernatant was taken to determine protein concentration using a BCA Protein Assay Kit (ZJ102, Shanghai Epizyme Biomedical Technology Co., Ltd), and the remaining supernatant was mixed with 5×SDS-PAGE sample loading buffer and kept in a 100 ℃ water bath for 10 min to denature its protein. Subsequently, we performed SDS-PAGE electrophoresis. Then the protein on the gel were electro-transferred to the PVDF membrane and blocked with 5% skimmed milk for two hours at room temperature. Then we washed off the excessive blocking solution with TBST buffer and incubated the PVDF membrane in the same TBST buffer with Nrf2, HO-1, Bax, Bcl-2, SOD-2 antibodies (1:1000; Cell Signaling Technology, USA); IL-1β, Tubulin (1:500; Santa, USA) at 4 ℃ overnight. The antibody recovered, the membrane was rinsed with TBST buffer on a 5-minute basis for three times and incubated with HRP-labeled secondary antibody for one hour. The protein expression was assessed by a chemiluminescence imager (Tanon 5200, YPHBIO, Beijing, China) with ECL Western Blotting Substrate (SparkJade, Shandong, China). The data was reviewed by Image J software which helps quantify the intensity of protein bands.

Quantitative real-time PCR

Take 10 mg of cardiac tissue (atria and ventricles) or approximately 2 × 10^6^ cardiomyocytes as a sample. TRIzol reagent (Solarbio biotechnology Co., Beijing, China) extracted 1 µg of total mRNA from samples as a template. The 1 st Strand cDNA Synthesis Super Mix kit (Yesen Co., Shanghai, China) converted total mRNA into cDNA by reverse transcription (20 µL system), and 1 µL of cDNA was taken for amplification. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) analysis was conducted on qPCR SYBR Green Master Mix. The reaction system was 20 µL. The primers (Supplemental Table S1; Supplemental Table S2) were synthesized and provided by Shanghai Biotechnology Co.

Histopathological examination

The heart fixed in 4% Paraformaldehyde was gradually dehydrated with alcohol of different concentration and transparentized with xylene. The tissues were lodged in paraffin, sliced into pieces of 4 μm each and stained with wheat germ agglutinin (WGA). Cardiac cells were scrutinized under a microscope for sizes.

To test ROS level, we conducted further verification on NRVMs. All three cell samples (CON, HG and CWP) were incubated with 2 µM Dihydroethidium (DHE, Beyotime Biotechnology, Shanghai, China) at 37 °C for 15 min. Images were acquired and studied by Image J. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) apoptosis detection kit (Promega Corporation, Madison, America) was applied in compliance with the manufacturer’s guidance to spot apoptosis. To assess cell surface area, immunofluorescent staining of F-actin (Beyotime Biotechnology, Shanghai, China, C2205S) was used following the manufacturer’s instructions.

Flow cytometry

H9C2 cells were subjected to various treatments and collected. The cell suspension was prepared according to Annexin V-FITC/PI Apoptosis Detection Kit (Yesen Co., Shanghai, China, 40302ES60). Next, it was allowed to react with Annexin V-FITC and propidium iodide dyes respectively in a dark environment and then examined with flow cytometry (BD Biosciences, New Jersey, America). The data analysis was conducted by FlowJo.

Statistical analysis

The outcome was demonstrated as means ± standard deviation (SD). Data were analyzed by one-way analysis of variance (ANOVA) and Tukey’s post-hoc test using GraphPad Prism 8.0. For all studies, values of p < 0.05 were considered statistically significant.

Results

Characteristics of the diabetic animal model and effect of CWP on body weight and heart weight

We monitored changes in body weight, blood glucose, food intake and water intake levels in all groups throughout the experimental period. One week after the intraperitoneal injection of STZ, the model group showed typical symptoms of type Ⅰ diabetes such as polydipsia, polyphagia, body weight loss, and increased blood glucose levels, confirming the successful establishment of the diabetic model (Table 1). Interestingly, in contrast with untreated subjects, the blood glucose of diabetic animals treated with CWP recovered to a certain level (Table 1). Additionally, the heart-to-body ratio of mice in the CON group was around 0.45 ± 0.02, which rose to 0.56 ± 0.11 in the DM group. But after CWP treatment, the ratio declined to 0.44 ± 0.04 (Table 1).

Table 1. Effects of different treatments on body weight, heart weight, heart body ratio, food uptake, water uptake, and random blood glucose levels in type Ⅰ diabetes miceTreatmentControlDMCWPFinal body weight (g)23.1 ± 3.120.1 ± 1.919.8 ± 1.3Absolut heart weight (g)0.12 ± 0.0130.1095 ± 0.02350.0945 ± 0.0145Heart body ratio (%)0.45 ± 0.020.56 ± 0.110.44 ± 0.04Food uptake (g) mus^− 1^day^− 1^energy (kcal) mus^− 1^day^− 1^4.15 ± 0.3512.53 ± 1.057.96 ± 1.19^^24.04 ± 3.59^^5.85 ± 0.8417.67 ± 2.54Water uptake (ml) mus^− 1^day^− 1^5.0 ± 0.39.9 ± 2.2^^7.8 ± 0.9Random blood glucose (mmol/L)8.51 ± 0.6120.66 ± 2.39^**^14.25 ± 1.92^#^CON, control group; DM, diabetic group; CWP, diabetic group treated with camel whey protein (CWP)Comparison between DM vs. control group: * P < 0.05, ** p < 0.01, *** p < 0.001Comparison between CWP groups vs. DM group: ^#^ P < 0.05

Efficacy on the vitality of serum enzymes concerning cardiac function

We tested some indicators of cardiac function and myocardial damage in mouse serum including CK-MB, CK, TG, LDL, AST and LDH. In contrast to the CON group, CK, TG and LDL in the DM group were higher by 43.5%, 71.4% and 33.1% respectively. After the CWP intervention these parameters were reduced by 6.2%, 73% and 23.5% respectively against the DM group. (Table 2).

Table 2. Effects of different treatments on cardiac function in type Ⅰ diabetes miceParameters estimatedControlDMCWPCK-MB (U L^− 1^)452.3 ± 63534.05 ± 64.45450.7 ± 70.7CK (U L^− 1^)1387.6 ± 411.81984.65 ± 203.05^^1861.8 ± 475.3TG (mmol l^− 1^)0.68 ± 0.251.54 ± 0.58^^0.415 ± 0.165^#^LDL (mmol l^− 1^)0.315 ± 0.0450.405 ± 0.015^^0.31 ± 0.03^#^AST (U L^− 1^)153.5 ± 25.8214.85 ± 32.25178.55 ± 40.45LDH (mmol l^− 1^)1327.8 ± 137.31565 ± 94.5^^1277.8 ± 181.5^#^CK-MB, creatine kinase isoenzyme; CK, creatine kinase; TG, triglyceride; LDL, Low density lipoprotein; AST, Aspartate aminotransferase; LDH, lactate dehydrogenaseComparison between DM vs. control group: * P < 0.05Comparison between CWP groups vs. DM group: ^#^ P < 0.05

CWP relieves myocardial hypertrophy in type I diabetes mice

Mice were administered according to the following procedure (Fig. 1A). To verify the efficacy of CWP on myocardial hypertrophy, WGA staining was performed on cardiac tissue sections to evaluate the morphology of mouse cardiomyocytes. In Fig. 1 (B, C) the volume of myocardial cells in the DM group notably exceeded that of the CON group. Owing to the effect of CWP, the volume of myocardial cells in the CWP group was remarkably smaller than that of DM group. In regard to transcriptional levels of Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), those in myocardial tissue of diabetic mice treated with CWP were greatly lower than those in the DM group (Fig. 1D, E).

Fig. 1. Effect of CWP on myocardial hypertrophy in type Ⅰ diabetes mice. (A) Timeline for in vivo experiments. (B-C) WGA staining and quantitative analysis of myocardial tissue. (D-E) RNA expression levels of ANP and BNP. STZ, streptozotocin; ANP, atrial natriuretic peptide; BNP, B-type natriuretic peptide. n = 6 mice per group. * p < 0.05, **** p < 0.0001

In conclusion, the experimental results indicated that CWP can mitigate myocardial hypertrophy caused by diabetes.

CWP inhibits the inflammatory reaction of myocardial tissue in type I diabetes mice

Inflammatory reaction is one of the main symptoms of DCM. We studied the influence of CWP on the inflammatory response of DCM. The mRNA expression of Interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the heart tissue of all groups were evaluated. The transcriptional level of inflammatory genes in the DM group were much higher than that of the CON group. As shown in (Fig. 2A-E) CWP normalized the elevated inflammatory genes due to DM. In the contrary the expression of Interleukin-10 (IL-10, an anti-inflammatory molecule) in the DM group was significantly reduced compared to CON group and CWP treatment enhanced its expression (Fig. 2F). At the protein level, pro-inflammatory genes such as 1β (IL-1β) was elevated in the DM group and CWP inhibited its up-regulation (Fig. 2G, H). In conclusion, CWP can protect cardiomyocytes from inflammatory reaction caused by hyperglycemia in diabetic mice.

Fig. 2. Effect of CWP on cardiac inflammatory response in type Ⅰ diabetes mice. (A-F) RNA expression level of IL-6, TNF-α, MCP-1, ICAM-1, VCAM-1, IL-10. (G-H) IL-1β immunoblot analysis and quantification. n = 6 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

CWP inhibits oxidative stress in myocardial tissue of type I diabetes mice by activatingNrf2/HO-1 signaling pathway

It has been confirmed that impaired antioxidative defensive ability causes DCM. In this research, we firstly tested the content of SOD and CAT in the serum samples of each group. Experimental data indicated that serum SOD and CAT activities decreased by 20.2% and 57.2%, respectively, in the DM group as compared to the CON group. SOD and CAT activities in the CWP group were found to increase by 29.2% and 126.3%, respectively, compared to the DM group (Fig. 3A, B). The same result was obtained in the test of the activity of SOD and CAT in tissues. Compared with the control group mice, the SOD and CAT activities in tissues of mice in the DM group decreased by 59.27% and 25.86%, respectively, while the SOD and CAT activities in the CWP group increased by 68.6% and 55.90% compared with the DM group (Fig. 3C, D). We also detected the protein expression level of SOD2 in the heart tissue from all groups. in comparison with the CON group, the expression level of SOD2 in the DM group apparently declined but evidently increased in the CWP group (Fig. 3E, G).

Fig. 3CWP inhibits oxidative stress in myocardial tissue of type I diabetes mice by activating Nrf2/HO-1 signaling pathway. (A-B) Activity of SOD and CAT in serum. (C-D) Activity of SOD and CAT in the heart tissues. (E-G) Western blotting and quantitative analysis of Nrf2, HO-1, SOD2 in the heart tissues. (H) RNA expression level of Nrf2 and HO-1 in the heart tissues. SOD, superoxide dismutase; CAT, catalase. n = 6 mice per group. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

Nrf2/HO-1 is an important signaling pathway associated with oxidative stress. We tested the protein and transcriptional levels of these two molecules in the heart tissue from all groups. Compared with the CON group, the protein levels of Nrf2 and its co-factor HO-1 were down-regulated in the DM group, but up-regulated with the CWP treatment (Fig. 3E, F). Same effects were also detected in transcriptional levels of them (Fig. 3H). In conclusion, CWP can inhibit the cardiac oxidative stress response caused by diabetes through Nrf2/HO-1 pathway. These results suggest the protective influence of CWP on the myocardium of diabetic mice is associated with the Nrf2/HO-1 signaling pathway, which has a significant effect on controlling oxidative stress.

CWP inhibits cardiomyocyte apoptosis in type I diabetes mice

The occurrence of cardiomyocyte apoptosis is among the factors that lead to cardiac structural deficiencies in diabetic patients. Therefore, we examined the ratio of two key proteins associated with apoptosis, namely Bcl-2 and Bax. Compared with the CON group, the ratio of Bax/Bcl-2 in the DM group was remarkably boosted while obviously declined in the CWP group with CWP treatment (Fig. 4A, B). The data suggests that CWP exert positive influence on the survival of cardiomyocytes in mice with diabetes.

Fig. 4. Effect of CWP on cardiomyocyte apoptosis. (A-B) Western blotting and quantitative analysis of Bax and Bcl-2. n = 4. * p < 0.05

CWP attenuated apoptosis and inflammatory responses in ventricular myocytes of newborn mice treated with high glucose

NRVMs were extracted to establish the ex vivo pathological trial in order to verify the preventive role that CWP might play in attenuating HG-induced damage to cardiac muscle cells. Firstly, according to our former research (Dou et al., 2022), different concentrations of CWP (i.e., 0.50, 1.00, 1.50 and 2.00 mg mL^− 1^) were utilized to intervene in the NRVMs, and the cell viability was analyzed by CCK8 test. The result showed that when the concentration was greater than 1.5 mg mL^− 1^, the CWP treatment induced significant growth inhibition on NRVMs (data not shown). So in the follow-up experiment, we took this concentration.

Cell apoptosis was measured by TUNEL staining as shown in Fig. 5 (A, B), which indicated that CWP treatment had decelerated cell apoptosis caused by HG stimulation. Meanwhile, HG stimulation up-regulated Bax and down-regulated Bcl-2 in HG group compared with controls, while those changes were reversed after CWP treatment (Fig. 5C, D).

Fig. 5. Effect of CWP on NRVMS apoptosis and inflammatory response. (A-B) Detection of apoptotic cells by TUNEL staining, positive cells were indicated by a white arrow and quantification of apoptotic cells. (C-D) Western blotting and quantitative analysis of Bax and Bcl-2. (E) RNA expression level of IL-6, TNF-α, MCP-1, ICAM-1, VCAM-1, IL-10. NRVMs, neonatal rat ventricular myocytes; HG, 33 mM glucose. n = 4. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

After that, we tested the level of pro-inflammatory cytokines, pro-inflammatory chemokines and cell adhesion molecules in different groups by qPCR. In consistence with the results of the in vitro, the transcriptional levels of IL-6, TNF-α, MCP-1, ICAM-1 and VCAM-1 were remarkably multiplied in HG group but decreased after CWP pre-treatment (Fig. 5E). We also detected the changes of antiphlogistic factor, IL-10, in different groups. We got the opposite trend compared to pro-inflammatory cytokines (Fig. 5E). In conclusion, the test results proved that CWP could protect NRVMs from apoptosis and inflammatory responses caused by HG stimulation.

We use H9C2 cell line to further verify the effect of CWP on cardiomyocyte apoptosis. Followed by Annexin V/PI staining, the percentage of apoptotic cells in HG group were greater than CON group and decreased after CWP treated. CWP treatment could slow down apoptosis induced by HG stimulation in cardiomyocyte (Sup Fig. 1 A).

CWP alleviated HG-Induced myocardial hypertrophy and oxidative stress in NRVMs

To determine whether CWP contributes to physiological myocardial hypertrophy, we detected the expression of ANP and BNP in different groups in vitro. We observed that the mRNA levels of ANP and BNP were both up-regulated in HG group in contrary to those of CON group. However, they were significantly down-regulated in CWP group in comparison with those of HG group (Fig. 6A). At the same time, we conducted F-actin immunofluorescence staining to test the effect of various treatments on the cell surface of NRVMs. It was found that the exposure of NRVMs cells to HG resulted in a significant increase in cell surface area while CWP attenuated such increase. (Fig. 6B, C).

Fig. 6. Effect of CWP on NRVMS myocardial hypertrophy and oxidative stress. (A) RNA expression levels of ANP and BNP. (B) Representative pictures of immunofluorescent staining for F-actin. (C) Quantification of cardiomyocyte size after CON、HG and CWP treatment. (D) RNA expression levels of Nrf2 and HO-1. (E) Superoxide dismutase was detected by DHE staining in NRVMs. (F-G) Western blotting and quantitative analysis of SOD2、Nrf2 and HO-1. n = 4. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001

It was assumed that uncontrolled increase in ROS output under high glucose condition would cause NRVMs dysfunction. Therefore, we clarified whether CWP performed an antioxidative role in HG stimulated NRVMs. ROS formation was boosted in HG stimulated NRVMs according to DHE staining while CWP pretreatment evidently reduced the expression of ROS (Fig. 6E). The anti-oxidant system under high glucose condition was modulated by Nrf2/HO-1 signaling pathway. The transcriptional levels of both Nrf2 and HO-1 were enhanced by CWP treatment (Fig. 6C, D). Western blotting results further proved that CWP maintained the high expression levels of Nrf2, HO-1 and SOD2 in response to HG challenges (Fig. 6F, G). The results proved that CWP could mitigate myocardial hypertrophy and oxidative stress injury induced by HG stress in NRMVs.

Discussion

Cardiomyopathy is a dangerous complication of diabetes, the final phase of which may witness heart failure, arrhythmia, cardiogenic shock and even sudden demise in serious cases. In recent years, with people’s health awareness strengthened, bioactive supplements such as camel milk have been more widely accepted. Our research has proved that CWP exerts a significant role in the prevention and the adjunctive treatment for DCM for the first time. Our findings suggest that CWP mitigates cardiac remodeling in diabetic mice by reducing myocardial hypertrophy, inflammation, oxidative stress and apoptosis. Containing many bioactive compounds that are vital to human health (Ho et al., 2022), camel milk is very similar to human milk in terms of nutritive value. It has been acknowledged that camel milk, especially the CWP which contains, has many biological functions such as reducing hyperglycemia caused by diabetes (Korish et al., 2020), enhancing immunosuppression and relieving oxidative stress induced by cyclophosphamide (Ibrahim et al., 2019). Furthermore, CWP can also facilitate the recovery of alcoholic liver in mice (Darwish et al., 2012).

The metabolic benefits of whey protein in blood glucose regulation and diabetes management arise from its dual regulatory effects on intestinal endocrine signaling cascades and amino acid-mediated pathways. Specifically, Camel whey protein exhibits multifaceted glycemic regulatory effects through its unique amino acid composition and hormonal interactions. Rich in branched-chain amino acids (BCAAs) and sulfur-containing amino acids, it rapidly stimulates the secretion of incretin hormones—glucose-dependent insulinotropic polypeptide (GIP) from duodenal K cells and glucagon-like peptide-1 (GLP-1) from ileal L cells—which enhance glucose-dependent insulin secretion while suppressing glucagon release (Drucker, 2018; Gribble & Reimann, 2021; Homayouni-Tabrizi et al., 2017; Korish, 2014; Wang et al., 2020). Concurrently, co-released cholecystokinin (CCK) and peptide YY (PYY) delay gastric emptying and promote satiety, collectively stabilizing postprandial glucose. The high leucine content acutely activates the mTOR/S6K1 axis in pancreatic β-cells to amplify insulin secretion, while isoleucine, valine, lysine, and threonine improve peripheral insulin sensitivity via AMPK activation and IRS-1 phosphorylation (Li et al., 2021; Yang et al., 2010). Furthermore, cysteine and methionine upregulate Nrf2-mediated antioxidant defenses and inhibit NF-κB-driven inflammation, protecting β-cell function from oxidative stress and mitigating diabetes progression. These synergistic mechanisms position camel whey protein as a potent dietary intervention for metabolic regulation (Brosnan & Brosnan, 2006; Djuric, 2018).

While the dosage of CWP applied to experimental mice was 400 mg/kg, the estimated dosage for human should be 400 mg/kg×3/36 = 33.33 mg/kg (Human dose = Animal dose× \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:\frac{\mathrm{A}\mathrm{n}\mathrm{i}\mathrm{m}\mathrm{a}\mathrm{l}\:\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}\:\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}}{\mathrm{H}\mathrm{u}\mathrm{m}\mathrm{a}\mathrm{n}\:\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{v}\mathrm{e}\mathrm{r}\mathrm{s}\mathrm{i}\mathrm{o}\mathrm{n}\:\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r}}$$\end{document} ,) (Zhang and Su, 1996). At the same time, because humans can consume 1–2 g/kg/day protein, humans can consume more CWP or camel milk, this may confer additional health benefits. However, this conversion method is based on theoretical estimation. To determine the practical dosage of CWP for human, clinical trials are required to verify its safety and effectiveness. In addition, factors such as metabolic differences in drugs from different species should be considered when conducting dose conversions in scientific research and drug development.

Normally, having gone through the gastric proteolytic degradation, CWP would enter the intestines and release peptide and/or amino acid molecules, which would be absorbed by the intestines and eventually transferred into blood circulation, exerting their biological and pharmacological effects on various target tissues. According to this study, it may play a role in fighting diabetic cardiomyopathy in heart tissue. However, some camel whey proteins are likely to resist proteolysis and stay as intact bioactive proteins (Anwar et al., 2021). As shown in many in vitro experiments, CWP as well as its derived hydrolysates can act directly or indirectly at the surface of cells and activate related intracellular signaling pathways to perform its biological functions. For example, CWP can reduce lipid accumulation and glycogen synthesis in HepG2 cells (Dou et al., 2022), activate insulin resistance in transfected HEK293 cells and native HepG2 cells (Khan et al., 2022), and promote cell proliferation of lymphocytes (Ebaid, 2014). CWP hydrolysates have positive effects on heat stress-induced rat liver BRL-3 A cells (Du et al., 2021). Some researches show that the CWP hydrolysates displayed significant antidiabetic activity compared with intact whey proteins (Kamal et al., 2018), which deserves further research in the future.

Cardiac dysfunction and ventricular structural deformation are the main features of DCM with myocardial hypertrophy being one of the major symptoms. We measured the heart-body ratio of all experimental objects and applied WGA staining to the myocardial cells. In coherence with earlier researches, myocardial hypertrophy occurred among all diabetic mice. However, the lesion was notably inhibited by CWP treatment. According to our test, the high expression levels of cordis hypertrophic signals namely ANP and BNP caused by hyperglycemia also declined remarkably due to CWP effect. These results suggested that CWP could alleviate cardiac hypertrophy in diabetic mice. Moreover, CWP reduced the expression of Bax and enhanced the expression of Bcl-2 in diabetic mice, which indicated that one of the mechanisms by which CWP protects the heart from DCM is to reduce cell apoptosis.

Studies have shown that DCM is closely related to myocarditis which has become a pathological process and can lead to cardiac hypertrophy and dysfunction (Frieler & Mortensen, 2015; He et al., 2022; Mann, 2015). Inflammation may disturb the process of myocardium metabolism and disrupt cardiomyocyte contractile properties. These abnormities jointly boost the progression of DCM (Frati et al., 2017). Earlier studies showed that camel milk can inhibit the expression of inflammation caused by colitis in mice (He et al., 2022). Some studies have pointed out that the antiphlogistic mechanism of camel milk is achieved by regulating inflammatory cells and inflammatory factors (Behrouz et al., 2022). This study found that CWP not only decreased pro-inflammatory cytokines (e.g. IL-6, TNF-α and IL-1β), pro-inflammatory chemokines (e.g. MCP-1) and cell adhesion molecules (such as ICAM-1 and VCAM-1) but also increased anti-inflammatory cytokines such as IL-10. It can be concluded that the preventive effect of CWP against hyperglycemia was achieved by attenuating inflammation in diabetic mice.

In addition to myocardial inflammation, oxidative stress is observed throughout the whole process of DCM. Oxidative stress disarranges post-translational modifications of contractive proteins in the early stage of DCM, which can induce myocardial structural distortion in exacerbated DCM (Waddingham et al., 2015). A majority of the pathogenic factors of DCM contribute to oxidative stress including hyperglycemia, hyperlipidemia, inflammatory cytokines and angiotensin system (Wu et al., 2019). Dysbolism induced by hyperglycemia stimulates the superabundant generation of ROS by mitochondrial electron-transport chain. Redundant ROS in diabetic cases is responsible for the progression of heart lesion. Being capable of removing free radicals, antioxidant enzymes (CAT, SOD and GPx) can perform as antioxidants (Hamed et al., 2018). According to previous studies, camel milk inhibits neurotoxic effects caused by fenpropathrin through mitigating oxidative stress, apoptotic, and inflammation in the mice’s brain (Abd-Elhakim et al., 2020). In this research, we detected the level of SOD and CAT in serum samples and the expression level of SOD2 in the heart tissue and NRVMs from all groups. The results showed that all the parameters mentioned above had been enhanced in the CWP group in contrast to the DM group, which means CWP could inhibit the oxidative stress response caused by DCM.

As a main transcriptional factor, Nrf2 adjusts the expression of endogenic antioxidant genes either directly or indirectly. Nrf2/HO-1 signaling pathway has also been widely noted as a main regulative pathway for endocellular protection against oxidative stress (Zhang et al., 2021). One study points out that vascular endothelial cells can be protected from oxidative stress by stimulating Nrf2/HO-1 pathway (Zhang et al., 2021). Many other researches have shown that the oxidative stress response can be modulated by Nrf2/HO-1 signaling pathway, which offers a method for curing pulmonary and bronchial diseases (Dang et al., 2020), renal ischemia-reperfusion injury (Diao et al., 2019) and acute pancreatitis (Liu et al., 2018). We examined the protein and mRNA levels of Nrf2/HO-1 in mouse heart tissue and NRVMs. Both results have proved that CWP can activate Nrf2/HO-1 signaling pathway to inhibit the oxidative stress response induced by DCM.

In summary, we have shown that CWP has a positive effect on diabetes cardiomyopathy. It can not only regulate cardiomyocyte morphology, inflammation and cardiomyocyte apoptosis but also activate Nrf2/HO-1 signaling pathway to mitigate oxidative stress. This study offers a new direction for the prophylaxis and treatment for DCM and broadens the prospect of CWP utilization.

Supplementary Information

Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary material 2 (DOCX 26.1 kb)