Application of Chlamydomonas reinhardtii red algae

Abstract

The present invention discloses an application of Chlamydomonas reinhardtii red algae. The Chlamydomonas reinhardtii red algae of the present invention can significantly reduce fasting blood sugar, body weight, triglycerides and low-density lipoprotein cholesterol, and also enhance oral glucose tolerance and insulin sensitivity. In pathological analysis, Chlamydomonas reinhardtii red algae significantly improved lipid deposition and liver tissue damage. Mechanistically, Chlamydomonas reinhardtii red algae can significantly reduce the expression of G‑6‑Pase and PEPCK proteins, regulate the SOCS2 / JAK2 / STAT5 signaling pathway, and improve gluconeogenesis.

Description

Technical Field

[0001] The invention relates to the technical field of food processing, and more particularly to an application of Chlamydomonas reinhardtii red algae. Background Art

[0002] Chronic metabolic disease diabetes is a disease characterized by persistent high blood sugar levels and is widely prevalent worldwide. Type 2 diabetes mellitus (T2DM) is the main form of diabetes, accounting for about 90% of all cases. According to a survey by the International Diabetes Federation, there are approximately 537 million people with diabetes aged 20-79 years worldwide.

[0003] Currently, a variety of chemical drugs have been used for the treatment of diabetes, such as metformin, sulfonylureas, α-glucosidase inhibitors, etc. However, these chemical drugs are inevitably accompanied by side effects. Therefore, the rich bioactive ingredients and non-toxic advantages of plants and algae have attracted more and more attention, making them a subject of increasing interest in diabetes treatment. In recent years, several studies have demonstrated the potential of green algae, spirulina, and brown algae supplements in improving symptoms related to type 2 diabetes and have the potential to reduce the use of medication.

[0004] Chlamydomonas reinhardtii green algae contains rich nutrients and active substances, including carbohydrates, lipids, polyunsaturated fatty acids, pigments and vitamins. Under various external environmental stress conditions such as ultraviolet radiation, temperature fluctuations, nutrient deficiency, increased salinity, and increased light intensity, the accumulation of certain nutrients will increase. According to reports, Chlamydomonas reinhardtii green algae has been found to have the potential to reduce fasting blood sugar levels and enhance immune response. And the study found that PPⅨ molecules play a vital role as an intermediate substance in the biosynthetic pathway of heme oxygenase (HO-1). HO-1 exerts an inhibitory effect on lipid peroxidation, reduces the production and accumulation of reactive oxygen species, and has a protective effect on the oxidative stress response of patients with T2DM.

[0005] Chlamydomonas reinhardtii red algae is produced by exposing Chlamydomonas reinhardtii green algae to ultraviolet radiation, resulting in increased accumulation of protoporphyrin IX (PP IX). Dried Chlamydomonas reinhardtii red algae contains about 5% protoporphyrin IX, and its color and flavor are comparable to meat. In addition, it has a high protein content and other bioactive ingredients, making it an ideal ingredient for the production of artificial meat. Adding only 0.3% can optimize the product's similarity to authentic meat, enhance its flavor characteristics, and reduce production costs.

[0006] However, the potential of the red alga C. reinhardtii to improve type 2 diabetes remains unclear. Summary of the invention

[0007] In view of this, the object of the present invention is to provide an application of Chlamydomonas reinhardtii red algae to solve the deficiencies in the prior art.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A Chlamydomonas reinhardtii (red) red algae, Chlamydomonas reinhardtii (red) TAI114, was purchased from Shanxi Touyun Biotechnology Co., Ltd. with a production batch number of 21121001.

[0010] A method for preparing the above-mentioned Chlamydomonas reinhardtii red algae specifically comprises the following steps: subjecting Chlamydomonas reinhardtii (obtained from the freshwater algae species bank of the Chinese Academy of Sciences, numbered FACHB-265) to ultraviolet light irradiation to obtain the obtained product.

[0011] Furthermore, the wavelength of the ultraviolet light irradiation is 200-380nm and the duration is 15s.

[0012] The present invention also claims to protect the use of the Chlamydomonas reinhardtii red algae in preparing health food for assisting in lowering blood sugar.

[0013] The present invention also claims to protect the use of the above-mentioned Chlamydomonas reinhardtii red algae in preparing health food for assisting in lowering blood lipids.

[0014] The present invention also claims to protect the use of the above-mentioned Chlamydomonas reinhardtii red algae in preparing a medicine for treating type 2 diabetes.

[0015] It can be seen from the above technical solution that compared with the prior art, the beneficial effects of the present invention are as follows:

[0016] The present invention discovers the effect of Chlamydomonas reinhardtii red algae rich in PPIX in improving T2DM and clarifies its potential molecular mechanism.

[0017] The Chlamydomonas reinhardtii red algae of the present invention can significantly reduce fasting blood sugar, body weight, triglycerides and low-density lipoprotein cholesterol, and also enhance oral glucose tolerance and insulin sensitivity. In pathological analysis, the Chlamydomonas reinhardtii red algae significantly improved lipid deposition and liver tissue damage. Mechanistically, the Chlamydomonas reinhardtii red algae can significantly reduce the expression of G-6-Pase and PEPCK proteins, regulate the SOCS2 / JAK2 / STAT5 signaling pathway, and improve gluconeogenesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The effects of the red alga Chlamydomonas reinhardtii on oral glucose tolerance and insulin resistance;

[0019] Figure 2 The effect of red algae Chlamydomonas reinhardtii on organ index;

[0020] Figure 3for the effects of the red alga Chlamydomonas reinhardtii on liver and pancreatic damage;

[0021] Figure 4 The effect of the red alga Chlamydomonas reinhardtii on inflammatory responses;

[0022] Figure 5 The effects of the red alga Chlamydomonas reinhardtii on blood lipids and liver oxidative stress;

[0023] Figure 6 Effects of the red alga Chlamydomonas reinhardtii on gluconeogenesis and the JAK2 / STAT5 pathway. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention are described clearly and completely below. 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 creative work are within the scope of protection of the present invention.

[0025] Example 1

[0026] 1 Materials and methods

[0027] 1.1 Animals, materials, and reagents

[0028] SPF 8-week-old male C57BL / 6 mice were purchased from the Experimental Animal Center of Hangzhou Medical College with a production license number of SCXK (Zhejiang) 2019-0002. During the experiment, the mice were kept in the barrier-level animal laboratory of Hangzhou Medical College with a use license number of SYXK (Zhejiang) 2016-0022. The breeding environment was a room temperature of 25±2°C, a relative humidity of 55±5%, and 12h light and dark alternation. The mice had free access to food and water and were adapted for 3 days before the experiment. The animal experiment was reviewed and approved by the Ethics Committee of Hangzhou Medical College and complied with the principles of animal protection.

[0029] Chlamydomonas reinhardtii (TAI114 strain) was purchased from Shanxi Touyun Biotechnology Co., Ltd., with production batch number 21121001. The strain was obtained by ultraviolet irradiation of Chlamydomonas reinhardtii (obtained from the freshwater algae seed bank of the Chinese Academy of Sciences, numbered FACHB-265). The composition of Chlamydomonas reinhardtii red algae includes 3%-7% protoporphyrin IX (PPIX), 30%-70% protein, fiber and carbohydrates, omega-3, 6 and 9 unsaturated fatty acids and essential vitamins and minerals.

[0030] Animal feed was purchased from Beijing Keao Xieli Feed Co., Ltd.; sodium citrate buffer solution was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; streptozotocin (STZ), glucose, insulin, hematoxylin-eosin (HE) (purity ≥ 98%), and oil red O staining (ORO) were purchased from Sigma-Aldrich Co., Ltd., USA; superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), malondialdehyde (MDA), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and total cholesterol (total cholesterol) were purchased from Sigma-Aldrich Co., Ltd., USA. The detection kits for cholesterol (TC) and triglyceride (TG) were purchased from Nanjing Jiancheng Bioengineering Institute; the detection kits for interferon-gamma (IFN-γ), interleukin-6 (IL-6), interleukin-1β (IL-1β), and monocyte chemotactic protein 1 (MCP-1) were purchased from Xinbosheng Biotechnology Co., Ltd.; the antibodies for SOCS2, JAK2, p-JAK2, STAT5, p-STAT5, and PEPCK were purchased from CellSignaling, USA; the antibody for G-6-Pase was purchased from Thermo Fisher Scientific, USA; the antibody for GAPDH was purchased from Hangzhou Baoke Biotechnology Co., Ltd.; the fluorescent secondary antibody was purchased from Licor, USA; the cDNA reverse transcription kit was purchased from Takara Biotechnology Co., Ltd.; the SYBR Green fluorescence quantitative PCR kit was purchased from Shanghai Yisheng Biotechnology Co., Ltd.; TRNzol total RNA extraction reagent was purchased from Beijing Tiangen Biochemical Technology Co., Ltd.

[0031] 1.2 Instruments and Equipment

[0032] A51119600DPC microplate reader; BA210 microscopy imaging system was purchased from Olympus, Japan; JA5003 electronic balance was purchased from Shanghai Sunny Hengping Scientific Instrument Co., Ltd.; Li-Cor Odyssey Clx two-color infrared laser imaging system was purchased from LI-COR, USA; NU-C200R-E low-temperature high-speed centrifuge was purchased from NUAIRE, USA; PowerPacTM HC protein electrophoresis transfer system was purchased from BIO-RAD, USA.

[0033] 1.3 Methods

[0034] 1.3.1 Animal model establishment and grouping

[0035] After a 5-day adaptation period, the mice were randomly divided into a high-fat diet (HFD) plus low-dose streptozotocin (STZ)-induced diabetes group (50 mice) and a control group (10 mice). To induce T2DM, 50 mice were fed a high-fat diet (XTHF60; Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd., containing 23% protein, 27% carbohydrates, and 35% fat) for 3 weeks. Subsequently, the mice were intraperitoneally injected with STZ (Sigma, St. Louis, MO, USA) at a dose of 35 mg / kg body weight (BW), twice, one week apart. Fasting blood glucose levels were measured one week after the last injection of STZ. Another 10 mice served as the control group and were fed a control diet (protein 19%, carbohydrate 61%, fat 5%; 1010009, Jiangsu Xietong Pharmaceutical Bioengineering Co., Ltd.). Then 50 mmol / L citric acid buffer was injected intraperitoneally. On the 7th day after the injection, the fasting blood glucose concentration of the mice in the treatment group was measured with a blood glucose meter (LifeScan, Milpitas, CA, USA). The model was successfully established when the fasting blood glucose concentration was ≥11.1 mmol / L. Chlamydomonas reinhardtii red algae was dissolved in 1% sodium carboxymethylcellulose solution. All HDF and STZ-induced diabetic mice were randomly divided into 4 groups: DM group, DM+CL group (low (L) concentration of Chlamydomonas reinhardtii red algae, 1g / kg BW / d), DM+CM group (medium (M) concentration of Chlamydomonas reinhardtii red algae, 2g / kg BW / d) and DM+CH group (high (H) concentration of Chlamydomonas reinhardtii red algae, 3g / kg BW / d). Mice were orally administered with Chlamydomonas reinhardtii red algae daily, and mice in the control group and DM group were orally administered with 1% sodium carboxymethylcellulose solution daily. All groups were orally administered for 4 consecutive weeks. The fasting blood glucose level was measured every two weeks to monitor the mice.

[0036] 1.3.2 Oral glucose tolerance test

[0037] At the end of the fourth week, mice were fasted for 16 h but not water, and gavaged with 2 g / kg BW glucose solution. Blood glucose concentration was measured with a blood glucose meter at 0, 15, 30, 60, and 120 min, and time-blood glucose concentration curves were plotted. The area under the curve (AUC) was calculated according to the following formula.

[0038] AUC = (0.5 × [H0 + H 30 ]+1.5×[H 30 +H 120 ]) / 2.

[0039] Where: H0, H 30 , H 120 It indicates the blood glucose concentration of mice at 0, 30 and 120 min (mmol / L).

[0040] 1.3.3 Insulin tolerance test

[0041] At the end of the fourth week, the mice were fasted for 4 h but not water, and insulin was injected intraperitoneally at 0.75 U / kg BW. The blood glucose concentration was measured with a blood glucose meter at 0, 15, 30, 60, and 120 min, and the time-blood glucose concentration curve was plotted to calculate the AUC.

[0042] 1.3.4 Blood biochemical test

[0043] At the end of the fourth week, mice were anesthetized with 2% 2,2,2-tribromoethanol, blood was collected from the medial canthal artery, and serum was collected after centrifugation at 4°C, 3000 r / min, and 15 min. The concentrations of blood lipid indicators TC, TG, LDL-C, and HDL-C were detected using a kit.

[0044] 1.3.5 Detection of inflammatory factors

[0045] The serum samples collected after centrifugation were used to detect the concentrations of inflammatory factor indicators IL-1β, IL-6, IFN-γ and MCP-1 using ELISA detection kits.

[0046] 1.3.6 Oxidation index detection

[0047] Accurately weigh the mouse liver tissue, add 9 volumes of normal saline according to the sample weight: normal saline volume = 1:9 (g:mL), prepare 10% tissue homogenate, centrifuge at 4°C, 3000 r / min, 10 min, and take the supernatant. Use the kit to detect SOD, GSH, CAT, and MDA activities.

[0048] 1.3.7 Histopathological observation

[0049] Mouse liver and pancreatic tissues were fixed with 10% formaldehyde solution, embedded in paraffin, sliced, stained with HE, sealed, and observed under a microscope at a magnification of 200 times.

[0050] 1.3.8 Observation of lipid deposition

[0051] Mouse liver sections were washed with isopropanol, stained with oil red dye, counterstained with hematoxylin, differentiated with ethanol, sealed, and observed under a microscope at a magnification of 400 times.

[0052] 1.3.9 Western blot detection

[0053] Western blot was used to quantify the protein expression levels of SOCS2, JAK2, p-JAK2, STAT5, p-STAT5, PEPCK, and G-6-Pase in liver tissue. 0.02 g of mouse liver tissue was taken and lysate was added to extract protein. The protein concentration was determined and separated, transferred to nitrocellulose membrane, incubated with primary antibody at 4°C overnight, and the results were analyzed using Odyssey 4.0.

[0054] 1.4 Statistical analysis

[0055] Data are expressed as mean ± standard deviation. SPSS22.0 software one-way analysis of variance (ANOVA) was used, and P < 0.05 was set as the standard for significant difference, which was statistically significant. GraphPad Prism 8 was used for plotting.

[0056] 2 Results

[0057] 2.1 Chlamydomonas reinhardtii improves oral glucose tolerance and insulin resistance

[0058] In order to study the regulatory effect of Chlamydomonas reinhardtii on metabolic disorders, we used Chlamydomonas reinhardtii to intervene in T2DM model mice for 4 weeks. The results showed that after 4 weeks of high-dose Chlamydomonas reinhardtii intervention, the fasting blood glucose (FBG) level and body weight of T2DM mice were significantly reduced ( Figure 1 Diabetic mice showed impaired oral glucose tolerance and insulin resistance, while intervention with the red alga Chlamydomonas reinhardtii significantly improved oral glucose tolerance and insulin resistance ( Figure 1 C and E). The area under the curve (AUC) results for oral glucose tolerance and insulin resistance were similar ( Figure 1 D and F).

[0059] 2.2 Effects of Chlamydomonas reinhardtii on organ indexes of diabetic mice

[0060] Effects of Chlamydomonas reinhardtii on organ indexes Figure 2 The results showed that the liver weight and adipose tissue weight of T2DM mice increased significantly, while the heart weight and kidney weight remained unchanged. However, intervention with Chlamydomonas reinhardtii effectively reduced the increase in liver weight in diabetic mice ( Figure 2 (A). Compared with normal mice, the liver and adipose tissue organ indexes of diabetic mice were significantly increased. However, intervention with the red alga Chlamydomonas reinhardtii did not improve these indexes ( Figure 2 E and F).

[0061] 2.3 Chlamydomonas reinhardtii reduces liver and pancreatic damage

[0062] H&E staining was used to perform histopathological examination of liver and pancreatic tissues of T2DM mice to evaluate the potential therapeutic effect of Chlamydomonas reinhardtii red algae intervention in T2DM mice. Figure 3 As shown in A, liver tissues of T2DM mice showed obvious hepatocyte vacuolation and lipid deposition, while intervention with Chlamydomonas reinhardtii red algae reduced hepatocyte vacuolation and fat droplets. Oil red staining showed that Chlamydomonas reinhardtii red algae effectively alleviated lipid accumulation in diabetic mice ( Figure 3 (B) The pancreas is an important organ responsible for the production of insulin. Figure 3 In C, the pancreatic islet structures in the control group were clearly visible and intact as shown by H&E staining, whereas in the diabetic group, the islets showed severe atrophy and disorganization, accompanied by a significant decrease in their number. The red algae intervention with Chlamydomonas reinhardtii significantly improved the damage to the islets, but did not completely restore them.

[0063] 2.4 Chlamydomonas reinhardtii reduces inflammatory response in diabetic mice

[0064] To elucidate the functional role of Chlamydomonas reinhardtii in the inflammatory response induced by T2DM, ELISA kits were used to quantify the levels of IL-1β, IL-6, IFN-γ, and MCP-1 in serum. The results showed that the levels of IL-1β, IL-6, IFN-γ, and MCP-1 in the serum of diabetic mice were significantly increased. It is worth noting that the intervention of Chlamydomonas reinhardtii effectively reduced the inflammatory effects of diabetic mice ( Figure 4 ).

[0065] 2.5 Effects of Chlamydomonas reinhardtii on blood lipids and liver oxidative stress

[0066] Figure 5 The results of the AD showed that fasting serum total cholesterol (TC), triglyceride (TG), and low-density lipoprotein cholesterol (LDL-C) levels were significantly increased, and high-density lipoprotein cholesterol (HDL-C) levels were significantly decreased in diabetic mice compared with control mice. After 4 weeks of treatment with Chlamydomonas reinhardtii red algae, TG and LDL-C levels in the high-dose group were significantly reduced compared with diabetic mice. However, Chlamydomonas reinhardtii red algae had no significant effect on TC and HDL-C levels.

[0067] The activities of superoxide dismutase (SOD), catalase (CAT) and reduced glutathione (GSH) in the liver tissue of diabetic mice were significantly inhibited, and the level of malondialdehyde (MDA) was significantly increased. Compared with untreated diabetic mice, oral administration of Chlamydomonas reinhardtii to diabetic mice resulted in a significant increase in CAT activity and GSH level ( Figure 5 F and G), while the MDA level in liver tissue decreased ( Figure 5 However, after treatment with Chlamydomonas reinhardtii, no significant changes in SOD activity were observed ( Figure 5 Middle E).

[0068] 2.6 Effects of Chlamydomonas reinhardtii on gluconeogenesis and the JAK2 / STAT5 pathway

[0069] The pathogenesis of T2DM is characterized by an imbalance in glucose homeostasis, leading to hyperglycemia. This imbalance is mainly caused by abnormal gluconeogenesis, and two enzymes, PEPCK and G-6-Pase, play a key role in regulating the gluconeogenesis process. In the liver tissue of diabetic mice, the expression levels of these key enzymes were upregulated. Oral administration of 2g / kg body weight / day and 3g / kg body weight / day of Chlamydomonas reinhardtii effectively reduced the levels of PEPCK and G-6-Pase ( Figure 6 A and B), indicating that it has the potential to downregulate the expression of these key enzymes and regulate hyperglycemia, thereby showing a protective effect against type 2 diabetes.

[0070] The SOCS2 / JAK2 / STAT5 signaling pathway regulates the expression levels of key genes related to gluconeogenesis. In the liver tissue of diabetic mice, the expression of SOCS2 was found to be downregulated, while treatment with the red alga Chlamydomonas reinhardtii resulted in the restoration of SOCS2 expression ( Figure 6 (C). Compared with the control group, the phosphorylation levels of JAK2 and STAT5 proteins in diabetic mice were significantly increased, while intervention with Chlamydomonas reinhardtii effectively reversed the levels of JAK2 and STAT5 phosphorylation ( Figure 6 (D and E). The red alga Chlamydomonas reinhardtii may regulate gluconeogenesis through SOCS2 by modulating the JAK2 / STAT5 signaling pathway.

[0071] 3 Conclusion

[0072] This study found that a red algae, Chlamydomonas reinhardtii, can significantly reduce fasting blood sugar, body weight, triglycerides and low-density lipoprotein cholesterol, while also enhancing oral glucose tolerance and insulin sensitivity. In pathological analysis, Chlamydomonas reinhardtii red algae significantly improved lipid deposition and liver tissue damage. Mechanistically, Chlamydomonas reinhardtii red algae can significantly reduce the expression of G-6-Pase and PEPCK proteins, regulate the SOCS2 / JAK2 / STAT5 signaling pathway, and improve gluconeogenesis.

[0073] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An application of Chlamydomonas reinhardtii red algae in preparing a health food that assists in lowering blood sugar.

2. An application of Chlamydomonas reinhardtii red algae in the preparation of health food that assists in lowering blood lipids.

3. Use of Chlamydomonas reinhardtii red algae in the preparation of a drug for treating type 2 diabetes.

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