Dangua Fang (丹瓜方) regulating tricarboxylic acid cycle and respiratory chain and its mechanism in diabetic rats

doi:10.19852/j.cnki.jtcm.20230904.002

Abstract

Abstract:

OBJECTIVE: To investigate the influence and possible targets of Dangua Fang (丹瓜方) on tricarboxylic acid (TCA) cycle and respiratory chain to enrich the prescription’s mechanism of effective intervention on glycolipid metabolic diseases such as type 2 diabetes.
METHODS: After interventional rats were fed with high glucose and high fat diet ad libitum for 4 weeks, intraperitoneally injected streptozotocin to induce diabetic model. According to blood glucose level,28 diabetic rats were selected and continued to be fed with high glucose and high fat diet, were stratified by body weight, and divided randomly by blood glucose into Model group (was given sterile water by gastric perfusion and injected aquae pro injection intraperitoneally), Dangua group [Dangua liquor (丹瓜方液) 20.5 g·kg^-1·d^-1 by perfusion and aquae pro injection intraperitoneally], Inhibitor group [sterile water by perfusion and nicotinamide phosphoribosyl transferase (Nampt) specific blocker GEN-617 1.25 mg/kg intraperitoneally], DanInhit group (Dangua liquor and GEN-617 synchronously). Control group were continuously fed with ordinary diet. The intervention was last for 10 weeks. Body weight (BW), liver index (LI), glycosylated hemoglobin (HbA1c), TC, TG, free fatty acids (FFA), creatinine (Cr), and A-ketoglutarate (α-KG), Iso-citric acid (ICA), oxaloacetic acid (OAA) were tested. The cytochrome C oxidase (COX) and Succinate dehydrogenase (SDH) were evaluated by Colorimetry; Nampt protein, Adenosine triphosphate (ATP) synthase (ATPs), Nicotinamide adenine dinucleotide (NAD⁺)and its reduced (NADH) in liver were measured by enzyme linked immunosorbent assay. The expressions of Nampt and mitochondrialnadhdehydrogenase-1 (mt-ND1) gene in liver was assessed by real-time polymerase chain reaction. Hepatic tissue staining was also completed.
RESULTS: The levels of BW, ICA, α-KG and Nampt-mRNA in the Model group are lower than that in the Normal group (P < 0.05), conversely, liver weight, LI, TC, HbA1c, SDH and ATPs, mt-ND1-mRNA, and Nampt protein in the Model group are higher (P < 0.01, P < 0.05). Compared with Model group, the levels of ICA, Nampt-mRNA and Nampt in Dangua group are significantly increased, and FFA obviously raised (P < 0.01 and P < 0.05); liver weight, BW, SDH are obviously lower, and HbA1c decreased significantly (P < 0.01, P < 0.05). TG, FFA and Nampt protein increased in the DanInhit group, TC, TG, BW obviously increased in the Inhibitor group, but SDH is decreased in both the two groups (P < 0.05, P < 0.01). Compared with Dangua group, DanInhib group has the lower levels of ICA, mt-ND1-mRNA, Nampt-mRNA, and the higher level of BW, LI and HbA1c. In the Inhibitor group, ICA and Nampt protein decreased, BW and LI, HbA1c and TG increased (P < 0.01 or P < 0.05). Tissue staining display that, in the model group there is obvious pathologic changes ie: fibrosis, steatosis and inflammatory cell infiltration. Lesions in the Dangua group are mild, and those of Inhibitor group are more obvious than the Model group, and DanInhit group is intermediately affected compared to Dangua group and Inhibitor group.
CONCLUSION: Dangua Fang increases the metabolic flux of TCA cycle and optimizes respiratory chain function by up-regulating Nampt expression.

Key words: Dangua Fang, diabetes mellitus, citric acid cycle, electron transport, nicotinamide phosphoribosyltransferase

HENG Xianpei, WANG Zhita, YANG Liuqing, LI Liang, HUANG Suping. Dangua Fang (丹瓜方) regulating tricarboxylic acid cycle and respiratory chain and its mechanism in diabetic rats[J]. Journal of Traditional Chinese Medicine, 2023, 43(6): 1150-1159.

Figures/Tables 7

Table 1 Tendency chart of weight change among groups ($\bar{x}$ ± s)

Group	n	Base	Week 2	Week 4	Week 6	Week 8	Week10
Control	7	487±30	554±39	572±36	579±35	591±34	585±33
Model	7	486±28	465±36^a	464±25^a	473±28^a	477±28^a	497±29^a
Dangua	7	489±23	447±27	472±25	491±27	483±22	509±24
DanInhibit	7	493±34	453±21	458±33	467±30	474±25	499±27
Inhibitor	7	492±34	498±38^b	499±35^c	506±23^c	514±34^c	525±34^c

Table 2 Liver weight and liver index among groups ($\bar{x}$ ± s)

Group	n	Liver weight (g)	Liver index (%)
Contol	7	14.13±1.46	2.41±0.15
Model	7	31.71±4.84^a	6.44±1.22^a
Dangua	7	25.18±4.84^b	4.95±0.90^b
DanInhib	7	33.48±4.97^c	6.88±1.69^c
Inhibitor	7	34.86±6.92^d	6.64±1.26^c

Figure 1 Comparison of HbA1c, TC, TG, FFA, and Cr A: percent of HbA1c; B: total cholesterol content; C: triglycerides content; D: free fatty acids content; E: serum creatinine content. Rats with normal blood glucose were fed with high-fat and high-sugar chow for 4 weeks, then injected with STZ 25 mg/kg for 2 consecutive days to selected the diabetic model. Control: fed a conventional diet without modelling; model: given sterile water by gastric perfusion once a day and injected aquae pro injection intraperitoneally every Monday at 3:00 pm; Dangua: Dangua liquor 20.5 g·kg-1·d-1 by perfusion and aquae pro injection intraperitoneally just like that in the model group; Inhibitor: sterile water by perfusion and GEN-617 intraperitoneally, 1.25 mg/kg; DanInhit: Dangua liquor and GEN-617 synchronously. STZ: streptozotocin; Cr: serum creatinine; TC: total cholesterol; TG: triglycerides; FFA: free fatty acids; HbA1c: glycosylated hemoglobin A1c. Data were expressed as the mean ± standard deviation (n = 7). Compared with control group, aP < 0.01, dP < 0.01; compared with model group, bP < 0.01, eP < 0.05; compared with Dangua group, cP < 0.01, fP < 0.05.

Figure 2 HE staining, Masson staining, and Oil red O staining of hepatic tissue (×100) A, D, G, J, M: HE staining (×100); B, E, H, K, N: Masson staining (×100); C, F, T, L, O: Oil red O staining (×100). A-C: Control group; D-F: model group; G-I: Dangua group: J-L: DanInhibit group: M-O: inhibitor group. Rats with normal blood glucose were fed with high-fat and high-sugar chow for 4 weeks, then injected with STZ 25 mg/kg for 2 consecutive days to selected the diabetic model. Control: fed a conventional diet without modelling (n = 7); model: given sterile water by gastric perfusion once a day and injected aquae pro injection intraperitoneally every Monday at 3:00 pm (n = 7); Dangua: Dangua liquor 20.5 g·kg-1·d-1 by perfusion and aquae pro injection intraperitoneally just like that in the model group (n = 7); Inhibitor: sterile water by perfusion and GEN-617 intraperitoneally, 1.25 mg/kg (n = 7); DanInhit: Dangua liquor and GEN-617 synchronously. Morphological samples were taken from the distal end of the hepatic right lobe (n = 7). HE: hematoxylin-eosin; STZ: streptozotocin.

Table 3 Comparison of OAA, ICA, α-KG, SDH, COX, and ATPs ($\bar{x}$ ± s)

Group	n	OAA (ng/μL)	ICA (ng/μL)	α-KG (ng/μL)	SDH (U/mg)	COX (ng/mL)	ATPs (pg/mL)
Contol	6	18.8±3.6	36.2±2.6	5.3±0.8	47.3±47.9	17.7±0.7	1406.9±108.6
Model	6	16.2±3.9	32.0±2.6^a	3.8±0.6^a	219.9±52.4^d	17.4±2.2	1563.6±126.4^a
Dangua	6	15.2±1.2	47.1±3.3^b	3.8±1.1	126.6±102.3^e	17.1±3.0	1506.3±125.7
Daninhib	6	17.3±3.2	32.3±3.9^c	4.4±1.6	116.2±94.8^e	18.1±2.3	1557.6±195.2
Inhibitor	6	15.8±3.6	30.4±4.2^c	4.1±1.7	54.4±46.7^b	18.3±1.5	1658.9±135.1^f

Figure 3 Melt curve plot and amplification plot of mt-ND1-mRNA and Nampt-mRNA A-C: amplification plot (n = 6). A: mt-ND1-mRNA; B: Nampt-mRNA; C: NADPH-mRNA. D-F: melt curve plot (n = 6). D: mt-ND1-mRNA; E: Nampt-mRNA; F: NADPH-mRNA. Rats with normal blood glucose were fed with high-fat and high-sugar chow for 4 weeks, then injected with STZ 25 mg/kg for 2 consecutive days to selected the diabetic model. Control: fed a conventional diet without modelling; model: given sterile water by gastric perfusion once a day and injected aquae pro injection intraperitoneally every Monday at 3:00 pm; Dangua: Dangua liquor 20.5 g·kg-1·d-1 by perfusion and aquae pro injection intraperitoneally just like that in the model group; Inhibitor: sterile water by perfusion and GEN-617 intraperitoneally,1.25 mg/kg; DanInhit: Dangua liquor and GEN-617 synchronously. NADPH as internal reference. It demonstrated that The Melt Curve Plot and Amplification Plot of mt-ND1-mRNA and Nampt-mRNA were comparable. mt-ND1: mitochondrial nadhdehydrogenase-1; Nampt: nicotinamide phosphoribosyltransferase; NADH: reduced NAD+; NAD+: nicotinamide adenine dinucleotide; STZ: streptozotocin.

Table 4 Comparison of mt-ND1-mRNA, Nampt-mRNA, Nampt, NAD+, NAD and NAD/NADH ($\bar{x}$ ± s)

Group	n	mt-ND1-mRNA	Nampt-mRNA	n	Nampt (ng/mL)	NAD⁺ (pg/mL)	NADH (pg/mL)	NAD/NADH
Contol	6	14.27±3.45	6.47±1.89	7	30.03±10.54	1158.26±239.45	395.72±53.84	2.93±0.49
Model	6	21.70±7.09^a	4.56±1.71^a	7	58.13±10.93^d	1256.68±217.25	429.53±81.82	2.99±0.62
Dangua	6	21.10±6.07	6.86±1.26^b	7	76.69±9.87^e	1284.05±259.42	405.87±52.72	3.23±0.79
DanInhib	6	18.82±4.31	4.60±1.21^c	7	72.57±13.39^b	1341.55±258.08	442.88±86.21	3.08±0.55
Inhibitor	6	18.80±5.75	5.40±1.70	7	62.46±4.10^c	1273.06±261.77	421.98±110.10	3.12±0.70

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