Effects of Coenzyme Q10 on Hepatic and Renal Injury and Bone in Diabetic Mice

 Uremia is a metabolic disorder characterized by chronic hyperglycemia due to insufficient insulin secretion or insulin resistance. Coenzyme Q10, a fat-soluble quinone, is a natural antioxidant that can resist damage caused by bacteria and free radicals and promote cell growth and self-repair [1]. Currently, in the field of diabetes mellitus, the research of coenzyme Q10 mainly focuses on the cardiovascular aspect of diabetes mellitus, and there are fewer reports on diabetes mellitus liver and kidney injury and bone. Therefore, in this study, we established a diabetes model by using streptozocin (STZ) to investigate the effects of coenzyme Q10 on liver and kidney injury and bone in diabetic mice.

 

Materials and methods

1 Material

Animals: 60 SPF Kunming mice, male and female, body mass 30-50 g, provided by Guangdong Medical Laboratory Animal Center. Animal Production License No.: SCXK (Guangdong) 2018-0002. This experiment was approved by the Ethics Committee of Guangdong Medical Animal Center (C202202-2).

 

Drugs and Reagents    

Metformin hydrochloride tablets, specification: 0.5 g per tablet, batch number: ABP0179, approved document number: National Drug Permit H20023370, produced by Shanghai Squibb Pharmaceuticals Co. STZ, purchased from Solembo Bioscience and Technology Co. Creatinine (Cr), blood urea nitrogen (BUN), total cholesterol (TC), triacylglycerides (TG), high density lipoprotein (HDL), low density lipoprotein (LDL), and high density lipoprotein (LDL) were obtained from Solebrite Biotechnology Co. LDL, Glutamic - Oxaloacetic Transaminase (GOT), Glutamic - Pyruvic Transaminase (GPT) kits were purchased from Ruixin Biological Company in Quanzhou, Fujian, China. Htoxylin eosin (HE) staining kit and Periodic Acid - Schiff (PAS) staining kit were purchased from Solepol Biotechnology Co.

 

Instrumentation   

Mini Bionix Material Testing System, product of MST, USA; V-1800 Visible Spectrophotometer, product of Avanti Instruments (Shanghai) Co., Ltd; BioTek Full Wavelength Enzyme Labeler, product of Shanghai Lu Xiangyi Centrifuge Instrument Co.

 

2 Experimental Methods

2.1 Establishment of STZ diabetic mouse model［2］

Mice were injected intraperitoneally with 50 mg kg-1 STZ 6 times, each time at an interval of 1 day. 2 weeks later, blood was collected from the tail vein of mice, and fasting blood glucose (FBG) was measured, and the fasting blood glucose (FBG) ≥ 11.1 mmol-L-1 was considered successful in the diabetes model. Fasting blood glucose (FBG) was measured in the tail vein of mice after 2 weeks.

 

2.2 Grouping and administration

Successfully modeled diabetic mice were randomly divided into 3 groups according to blood glucose: the model group (n = 20), the metformin group (n = 10), and the coenzyme Q10 group (n = 10), with each group divided into 50/50 males and females. The model group was given saline by gavage; the metformin group was given 500 mg kg-1 d-1 metformin hydrochloride; the coenzyme Q10 group was given 3 000 mg kg-1 d-1 coenzyme Q10; and the normal group consisted of 20 normal mice with equal amounts of saline by gavage. The normal group consisted of 20 normal mice given saline by gavage once a day for 75 days.

 

2.3 Indicator testing

2. 3. 1 Fasting blood glucose test with glucometer

Fasting blood glucose was measured every 10 days by blood sampling from the tail vein with a glucometer, and the mice were fasted for 12 h before each measurement.

 

2. 3. 2 Detection of serum biochemical indices by spectrophotometer and enzyme marker

Level [3]

On the 76th day, mice were killed by blood sampling from the eyeballs, blood was collected and centrifuged at 2,000 r-min-1 for 10 min at 4 ℃, and the supernatant was stored at -80 ℃ for reserve. Take an appropriate amount of serum and dilute it according to the actual situation and the instructions in the manual. According to the instructions of the kit, use the sarcosine oxidase method to determine Cr, the urease method to determine BUN, the Rai method to determine GOT and GPT, the cholesterol oxidase method to determine TC, the phosphoglycerol oxidase method to determine TG, and the direct method to determine HDL and LDL, and then finally obtain the optical density values of the indexes, and then according to the relevant standard curve, the corresponding indexes are obtained by substituting the various values into the standard curve. According to the standard curve, the values of each index can be substituted to obtain the serum concentration of the corresponding index.

 

2. 3. 3 Determination of bone biomechanical parameters by three-point bending tests [4]

After drug administration, the femur bone of mice was removed from the bone and stored in 70% ethanol for bone biomechanical examination. The 858 Mini Bionix Material Testing System (MTS) was used to perform three-point bending test to analyze the biomechanical properties of the femur. The mouse femur was placed on the MTS machine and loaded at 0.155 mm - s - 1 , with a loading speed of 0.5 mm - s - 1 . 155 mm-s-1 with a span of 5 mm. The load-deformation curves were plotted, read from the curves, and the parametric indexes were calculated according to the corresponding formulae: maximum load, break load, elastic load, stiffness.

 

2. 3. 4 Observation of histopathological changes in kidney and liver tissue of mice by HE staining and PAS staining [5-6].

After drug administration, kidney and liver tissues were taken from mice and fixed with 4% paraformaldehyde, then dehydrated with gradient concentration of ethanol, permeabilized with xylene, and embedded with paraffin, then 4 μm thick tissue sections were made, which were de-waxed, immersed in water, stained, dehydrated, made transparent, and sealed for histopathological observation.

 

3 Statistical processing

Data were statistically analyzed using SPSS 26.0 software. Measurements were expressed as x- ± s. The t-test was used to test for chi-square, and the corrected t-test was used to test for unequal variances.

 

Results

1 Changes in blood glucose in each group of mice

The mice were randomly divided into the normal group (saline gavage), the model group (diabetes model), the metformin group (500 mg - kg-1 - d-1 metformin hydrochloride) and the coenzyme Q10 group (3 000 mg - kg-1 - d-1 coenzyme Q10). The fasting blood glucose results showed that the mice in the model group, metformin group and coenzyme Q10 group had significantly higher fasting blood glucose compared with those in the normal group, and the differences were statistically significant (P < 0.05); the fasting blood glucose of the mice in the metformin group and the model group decreased significantly on the 30th day, and the differences were statistically significant (P < 0.05); the mice in the other days showed some decreases, but the differences were not statistically significant (P > 0.05). ＞The difference was statistically significant (P<0.05). In the coenzyme Q10 group, there was a statistically significant decrease in fasting blood glucose on day 50, and the difference was statistically significant (P<0.05). The results are shown in Table 1.

 

2 Effect of coenzyme Q10 on serum liver and kidney function indices in diabetic mice

Compared with the normal group, mice in the model group, metformin group and coenzyme Q10 group showed significantly higher levels of BUN and Cr, which are indicators of renal function, and GPT and GOT, which are indicators of hepatic function, and the differences were statistically significant (all P<0.05). Compared with the model group, the serum levels of BUN, GPT and GOT were significantly lower in the metformin group, and the serum levels of BUN, Cr, GPT and GOT were significantly lower in the coenzyme Q 10 group, with statistically significant differences (all P<0.05), indicating that there were serious liver and kidney injuries. The differences were statistically significant (all P<0.05), indicating that the liver and kidney injuries were improved in the metformin and coenzyme Q 10 groups. The results are shown in Table 2.

 

3 Effect of coenzyme Q10 on serum lipid indices in diabetic mice

In the model group and metformin group, compared with the normal group, serum lipid indices TC, TG and LDL were significantly increased, and HDL was significantly decreased, with statistically significant differences (all P＜0.01); in the coenzyme Q10 group, compared with the normal group, serum TC, TG and LDL were significantly increased, with statistically significant differences (all P＜0.01); in the metformin group and coenzyme Q10 group, compared with the model group, serum TC, TG and LDL were significantly decreased, with statistically significant differences (all P＜0.01). In the metformin group and coenzyme Q10 group, serum TC, TG and LDL were significantly lower than those in the model group, and the differences were statistically significant (all P＜0.01); in the coenzyme Q10 group, HDL was significantly higher than those in the model group, and the differences were statistically significant (P＜0.01). The results are shown in Table 3.

 

4 Changes in bone biomechanical parameters in each group

In the model group and coenzyme Q10 group, compared with the normal group, the maximal load and fracture load were significantly reduced, and the differences were statistically significant (all P＜0. 05), and the rigidity coefficient decreased, but the differences were not statistically significant (P＞0. 05); in the metformin group, compared with the model group, the maximal load and fracture load of the femur increased, and the differences were statistically significant (all P＜0. 05), and the maximal load, fracture load, and stiffness coefficient tended to rise, but were not statistically significant (P＞0.05). The elastic load, fracture load and stiffness coefficient tended to increase, but the differences were not statistically significant (P＞0.05), while the maximal load and fracture load increased and the stiffness coefficient decreased in the coenzyme Q10 group, but the differences were not statistically significant (P＞0.05). The results are shown in Table 4.

 

5 Changes in renal organization of mice in each group

HE staining and PAS staining showed that the normal group mice had normal renal tissue structure, regular glomerular morphology, no proliferation of glomerular mesangial cells, and no thickening of glomerular basement membrane; compared with the normal group, the model group mice had abnormal renal tissue structure, proliferation of glomerular mesangial cells, thickening of glomerular basement membrane, and focally infiltrated inflammatory cells and a small amount of fibrotic tissue hyperplasia in interstitium; in comparison with the model group, the Metformin and Coenzyme Q10 groups showed improvement in glomerular mesangial cell proliferation and thickening of the basement membrane. Compared with the model group, the metformin and coenzyme Q10 groups showed improved glomerular mesangial cell proliferation and basement membrane thickening. The results are shown in Figure 1.

 

6 Changes in liver tissue structure of mice in each group

HE staining showed that the hepatocytes in the normal group were arranged in a single radial row centered on the central vein; compared with the normal group, the hepatocytes around the central vein in the model group showed vacuolated degeneration; compared with the model group, the hepatocytes in the metformin group and the coenzyme Q10 group were arranged in a more regular manner, and the vacuolated lesions were ameliorated. The results are shown in Figure 2.

 

discussion

Compared with the normal group, the mice in the model group showed a significant increase in fasting blood glucose, significant changes in liver and kidney function indexes, blood lipid indexes, and a significant decrease in bone biomechanical parameters, suggesting that the mouse diabetes model was successful and accompanied by liver and kidney damage as well as a decrease in bone hardness.

Studies have shown that coenzyme Q10 can protect against hepatotoxicity induced by antituberculosis therapy by reducing oxidative stress and inflammatory damage [7], and can also improve immunosuppressant-induced renal mitochondrial dysfunction by reducing oxidative stress [8]. The results of the present experiment showed that the fracture load and maximum load of femur in coenzyme Q10 mice increased compared with the model group, and Q10 could improve the bone biomechanical properties of ovariectomized rats. Therefore, coenzyme Q10 may improve bone injury in diabetic mice [9].

 

The results showed that repeated low-dose intraperitoneal injections of 50 mg kg-1 STZ into mice for 6 times with an interval of 1 d each could destroy the pancreas, cause elevation of blood glucose, and establish a diabetic model in mice, as well as cause damage to the liver and kidneys, and decrease the hardness of the bones. The innovation of this study is that repeated low-dose intraperitoneal injections of STZ were used to establish a diabetic mouse model, which has a high survival rate and a high success rate, and it is the first time to observe the effects of coenzyme Q10 on the kidney, liver, and bone of diabetic mice, which suggests that coenzyme Q10 has a protective effect on the liver and kidney, and may have a positive effect on the bone, which can provide experimental evidence for the prevention of clinical treatment of diabetes mellitus and the damage to the liver and kidney.

 

References:

［1］ WANG Hongling, WU Tingru, ZHANG Xiumei.  Research progress on antioxidant property and application of coenzyme Q10 ［J］.  Food Research and Development, 2015, 36(19): 188 - 190.

［2］ Zhao Miaoxin.  Study on the protective effect of saffron yellow pigment on the kidney of mice with diabetic nephropathy and related mechanism［D］.  Liaoning Jinzhou: Jinzhou Medical University, 2021.

［3］ Chen Xuelin, Hu Jianzhuo.  Effects of Quenching Thirst Formula on Glycolipid Metabolism and Liver and Kidney Injury in Diabetic Rats［J］.   Sichuan Traditional Chinese Medicine, 2021 , 39(4):50-53.

［4］ Wang Kexin, Chen Cailing, Zheng Xiaoyan, et al.  Effects of coenzyme Q10 on bone microstructure and myogenic fibers in D - galactose-induced male mice [J].  Chinese Pharmacology Bulletin, 2019, 35(11):1544 - 1550.

［5］ TANG Hui, YAO Zhihao, LUO Daowen, et al.  Establishment of a rat model of type 2 diabetic osteoporosis by combining high-fat and high-sugar diet with streptozotocin［ J］.   Chinese Tissue Engineering Research, 2021 , 25(8):1207 - 1211 .

［6］ Zhang Meng, Yang Licheng, Chen Juan, et al.  Protective effects of polysaccharide fractions from peony bark on renal injury in rats with diabetic nephropathy［J］.  Chinese Journal of Traditional Chinese Medicine, 2022, 47(3):713-720.

［7］ BASKARAN U L, SABINA E P. The food supplement coenzyme Q10 and suppression of antitubercular drug - induced hepatic injury in rats: the role of antioxidant defense system, anti - inflammatory cyto - kine IL - 10 [J].   Cell Biol Toxicol, 2015, 31(4 -5): 211 -219.

［8］ YU J H, LIM S W, LUO K, et al. Coenzyme Q10 alleviates tacroli- mus - induced mitochondrial dysfunction in kidney［J］.  faseb j, 2019, 33(11):12288 - 12298.

［9］ Chen Lisi, Huang Zhirong, Wu Haiyou, et al.  Effects of coenzyme Q10 on microstructure and biomechanics of femur in de-ovulated rats [J].  Chinese Journal of Osteoporosis, 2016, 22(8):944 -950.