Cistanche Deserticola Polysaccharides Attenuates Diabetic Nephropathy in Mice By Affecting The Intestinal Flora And Inhibiting The Toll-Like Receptor 4/Nuclear Factor-κB Signaling Pathway

Abstract: The objective of this study was to investigate the ameliorative effect and potential mechanism of Cistanche deserticola polysaccharides (CDPs) on diabetic nephropathy (DN) in mice. The DN mouse model was established by high fat diet (HFD) feeding combined with an intraperitoneal injection of streptozotocin (STZ). Thirty-six C57BL/6N male mice were randomly divided into six groups (n = 6 each). All groups were orally administered with distilled water (control and model), dapagliflozin or CDPs for 4 weeks. Changes in physiological indices, renal function, inflammatory factors and the intestinal microbiota were assessed. The results showed that CDPs significantly improved the general condition and attenuated the renal histopathological changes, and reduced the levels of blood glucose, urinary protein and inflammatory factors in DN mice. Additionally, CDPs affected the balance of intestinal microbiota, increased the abundance of beneficial bacteria and decreased potential pathogenic bacteria, which in turn improved intestinal barrier function. CDPs also inhibited the activation of the Toll-like receptor 4/nuclear factor-κB (TLR4/NF-κB) signaling pathway and reduced the release of lipopolysaccharide (LPS). In conclusion, CDPs exhibit an ameliorative effect on DN in mice by affecting the intestinal microbiota and inhibiting inflammatory signaling pathways. This finding offers new insights into the treatment of DN.

 

Keywords: Cistanche deserticola polysaccharides; diabetic nephropathy; intestinal barrier; intestinal flora; Toll-like receptor 4; nuclear factor-κB

 

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Diabetes nephropathy (DN) is the leading cause of end-stage renal disease and seriously threatens the life and health of patients[1]. Studies have confirmed that intestinal flora imbalance plays an important pathogenic role in the process of diabetes developing into DN[2-3]. The "gut-kidney axis" theory believes that changes in the intestinal microenvironment can affect the progression of chronic kidney disease by directly regulating the metabolites of the intestinal flora[2-3]. The intestinal flora plays an important role in regulating renal function in DN mouse models[4-5]. In-depth research on the "gut-kidney axis" will help clarify the pathogenesis of DN and discover new targets for prevention and treatment.
Cistanche deserticola is a traditional medicinal and edible plant, also known as Cistanche deserticola, Dijing, Jinsun, etc., commonly known as Dayun, and has the reputation of "desert ginseng"[6]. According to the Pharmacopoeia of the People's Republic of China, Cistanche deserticola is the dried fleshy stem with scaly leaves of Cistanche deserticola Y. C. Ma or C. tubulosa (Schenk) Wight, a plant of the genus Cistanche in the family Orobanchaceae.
Among them, Cistanche deserticola has a long history of being used as both medicine and food. Cistanche deserticola is sweet, salty, and warm in nature and enters the kidney and large intestine meridians. It has the effects of tonifying kidney yang, benefiting essence and blood, and moistening the intestines and relieving constipation [7-8]. Polysaccharides are one of the main active ingredients of Cistanche deserticola. The content of polysaccharides in Cistanche deserticola water extract is relatively high. It is mainly composed of monosaccharides such as glucose, galactose, rhamnose, arabinose, and fructose. It has many pharmacological effects such as regulating immune activity, anti-aging, improving learning and memory, protecting nerves, resisting liver damage, anti-virus, and anti-tumor [9-12]. Previous studies by the research group have shown that Cistanche deserticola water extract has a potential therapeutic effect on DN [13]. Based on this, this experiment took the intestinal flora and inflammatory response as the starting point to study the effect of Cistanche deserticola polysaccharides (CDPs) on improving DN in mice and restoring kidney function, in order to provide a scientific basis for the role of CDPs in treating DN.

1 Materials and methods

1.1 Animals, materials and reagents

36 6-week-old C57BL/6J male mice were purchased from the Animal Experiment Center of Xinjiang Medical University, Animal Production License No.: SCXK (Xin) 2023-0006; Ethics Resolution No.: IACUC-20210331-04.
Desert CDPs (polysaccharide mass fraction ＞80%, batch number: 2306019) Shaanxi Xintianyu Company; dapagliflozin (Dapa; batch number: 2108119) AstraZeneca Pharmaceuticals Co., Ltd.; urine total protein (urine total protein, UTP; batch number: C035-2-1), urea nitrogen (blood urea nitrogen, BUN; batch number: C013-2-1), creatinine (creatinine, Cre; batch number: C011-2-1), uric acid (uricacid, UA; batch number: C012-2-1) kit Nanjing Jiancheng Bioengineering Institute; lipopolysaccharide (LPS; batch number: YX-121619M), interleukin-6 (IL-6; batch number: YX-E20012), IL-1β (batch number: YX-E20533), transforming growth factor-β (TGF-β; batch number: YX-E20217), tumor necrosis factor-α (TNF-α; batch number: YX-E20220) kit Shanghai Youxuan Biological Co., Ltd.; enhanced chemiluminescene (ECL) kit Beijing Pulilai Gene Technology Co., Ltd.; streptozotocin (STZ; batch number: 20230503), high-fat diet (60% fat ratio high-fat diet purified type, material number: Boaigang12492M) Beijing Boaigang Biotechnology Co., Ltd.
The basic feed (containing 22% corn flour, 12% bran, 18% oil residue, 8% fish meal, 12% soybean meal, 0.17% salt, 0.25% vitamins, 0.25% trace elements, 0.33% cod liver oil, and 2% stone powder) was provided by the Animal Experiment Center of Xinjiang Medical University.

 

1.2 Instruments and equipment

AE240 analytical balance, Mettler-Toledo Instrument Co., Ltd., Switzerland; DHG-9053A electric heating blast drying oven, Shanghai Yiheng Scientific Instrument Co., Ltd.; DK-S22 constant temperature water bath, Shanghai Jinghong Experimental Instrument Co., Ltd.; 3-18KS high-speed low-temperature centrifuge, SigmaAldrich, USA; BS-380 fully automatic biochemical analyzer, Shenzhen Mindray Biomedical Electronics Co., Ltd.; EM KMR3 slicer, HI1220 slicer, Leitz Optilux digital microscope, Leica, Germany; FluorChem E chemiluminescence imaging system, ProteinSimple, USA.

1.3 Methods

1.3.1 Animal experiment

36 C57BL/6J male mice were fed in the SPF laboratory of Xinjiang Medical University and fed freely. The experiment was carried out after 7 days of adaptive feeding. Six mice were randomly selected as the normal (Control) group and given a basic diet. The rest of the experimental groups were fed a high-fat diet and kept in separate cages. The body weight was tested once a week. The mice were free to eat and drink water. After 6 weeks of high-fat diet feeding, the experimental group mice were injected with STZ 40 mg/kg intraperitoneally for 5 consecutive days in addition to free high-fat diet every day. The fasting blood glucose after modeling was 11.1 mmol/L, which was considered a successful model of diabetes. Thirty mice with successful modeling were randomly divided into five groups, with six mice in each group, namely the model (distilled water + high-fat diet, Model) group, Dapa (Dapa 4 mg/kg + high-fat diet, Dapa) group, CDPs low-dose (50 mg/kg + high-fat diet, CDPs-L) group, CDPs medium-dose (100 mg/kg + high-fat diet, CDPs-M) group, and CDPs high-dose (200 mg/kg + high-fat diet, CDPs-H) group. The control group (distilled water + normal diet) was given an equal amount of distilled water. Each mouse was gavaged at 0.1 mL/10 g, once a day at the same time, for a total of 4 weeks. The body weight and blood glucose changes of the mice were recorded weekly.

1.3.2 Index determination

After 4 weeks of gavage administration, urine samples (24 h) and fresh feces were collected through metabolic cages at the end of the 4th week and stored at -20 ℃ and -80 ℃, respectively. The mice were fasted but not watered for 12 h. The next day, the fasting body weight of the mice was measured. The mice were anesthetized by intraperitoneal injection of 3 mL/kg of 10% chloral hydrate solution. Blood was collected from the orbits and centrifuged in a centrifuge (4 °C, 3 500 r/min) for 15 min. The supernatant was aspirated, packaged, and stored in a -80 °C refrigerator for later use. The serum was measured for BUN, Cre, UA, IL-6, IL-1β, TGF-β, TNF-α, and LPS according to the kit. The urine was measured for 24 h UTP according to the kit.

 

1.3.3 Morphological and pathological observation of kidney and intestine

1.3.3.1 Determination of Kidney Coefficient

After blood collection, mice were anesthetized to death by overdose, and the kidneys and colons were completely removed and immediately rinsed with pre-cooled saline, drained on filter paper and weighed. The kidney coefficient was calculated according to the following formula: Kidney coefficient/%= ×100 m1m, where m1 is the wet weight of kidney/g; m is the body weight of mouse/g.

 

1.3.3.2 Pathological observation of kidney

Mouse kidneys were fixed in 4% paraformaldehyde fixative, dehydrated by ethanol solution gradient, transparentized by xylene, embedded in paraffin, sliced ​​(thickness 2 μm), dewaxed by xylene, and hydrated by ethanol solution gradient, and then stained with hematoxylin & eosin (H&E), Masson, and periodic acid-Schiff (PAS), sealed, and images were collected and observed under a microscope.

 

1.3.3.3 Intestinal pathological observation

The mouse intestine was fixed with 4% paraformaldehyde fixative, dehydrated with ethanol solution gradient, transparentized with xylene, embedded in paraffin, sliced ​​(thickness 2 μm), dewaxed with xylene, and hydrated with ethanol gradient, then stained with H&E and Alcian blue (AB)-PAS for sealing, and images were collected under a microscope for observation.

 

 

1.3.4 Analysis of intestinal flora composition

The colon contents of mice in the Control group, Model group, and CDPs-H group were detected by 16S rRNA gene high-throughput sequencing. The DNA extraction and library construction and sequencing of microorganisms in the colon contents were completed by Lianchuan Biotechnology Co., Ltd., and the obtained data were analyzed on the Lianchuan Cloud Platform.

 

1.3.5 Detection of kidney and colon-related protein expression

The mouse kidney and colon tissue proteins were extracted, and the protein was quantified by the bicinchoninic acid protein quantification method. After protein denaturation, the protein was stored in a -20 ℃ refrigerator. 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polyvinylidene difluoride membrane transfer, the membrane was placed in a tris buffered saline with Tween-20 (TBST) incubation tank, and blocked at room temperature. Detection
The primary antibodies to Toll-like receptors (TLR) 4, nuclear transcription factor (NF)-κB p65, p-NF-κBp65, and β-actin in kidney tissue (antibody diluted 1:2 000 (V/V) with TBST solution) and occludin and zonula occludens-1 (ZO-1) in colon tissue (antibody diluted 1:1 000 (V/V) with TBST solution) were incubated at 4 ℃ overnight. TBST dilution, secondary antibody (antibody
diluted with TBST solution 1∶2 000 (V/V)), incubated at room temperature for 1 h, washed with TBST 3 times, and then detected by adding ECL luminescent reagent, and gel imaging was used to detect protein expression. β-actin was used as an internal reference, and the protein gray value was analyzed by ImageJ software. The protein expression of each group was expressed as the relative value of the gray value of the control group.

 

1.4 Data statistics

All data results were expressed as ±s, and SPSS 21.0 was used for statistical analysis. For continuous data, if they followed a normal distribution, one-way analysis of variance was used for inter-group comparison. If the difference between the groups was statistically significant, the Turkey method was further used for pairwise comparison; if the data followed a normal distribution, the Kruskal-Wallis H test was used for inter-group comparison. When there was a statistical difference between the groups, Durbin's least significant difference method was further used for multiple comparisons. P < 0.05 indicated that the difference was statistically significant.

 

 

2 Results and analysis

2.1 Effect of CDPs on blood glucose, body weight, and kidney coefficient of C57BL/6J

As can be seen from Tables 1 to 3, compared with the Control group, the fasting blood glucose of mice in the Model group was significantly increased (P<0.01), and the kidney coefficient was significantly increased (P<0.01); compared with the Model group, CDPs- The body weight of mice in group H increased significantly after 4 weeks of administration (P＜0.05), and blood sugar began to decrease after 3 weeks of administration (P＜0.05). The kidney coefficient of mice in each intervention group decreased, and the CDPs-H group was the lowest (P<0.01); the body weight of mice in the Control group was larger than that of each group, indicating that CDPs can slow down the swelling of the kidneys of DN mice, thereby Reduce the kidney coefficient.

 

Table 1 Effects of CDPs on blood glucose of mice (n = 6)

Group	Time/Week
	0	1	2	3	4
Control	7.98±0.55	7.32±0.40	7.58±0.50	7.38±0.87	7.28±0.34
Model	10.62±2.79#	20.32±2.69#	24.47±4.18#	24.92±3.98#	20.33±2.96#
CDPs-L	16.03±2.97	21.83±4.81	20.13±6.97	23.30±3.77	21.25±1.65
CDPs-M	13.17±3.59	22.6±5.88	19.35±4.45	22.6±4.87	18.38±1.20
CDPs-H	11.07±1.74	21.53±3.71	21.57±6.19	17.43±2.05**	15.87±0.90**
Dapa	10.28±4.75	21.12±4.96	17.72±2.00**	17.35±2.00**	14.55±1.06**

Unit: mmol/L

 

Table 2 Effects of CDPs on kidney index of mice (n = 6)

 

Group	Control	Model	CDPs-L	CDPs-M	CDPs-H	Dapa
Kidney/Body Weight %	0.98±0.04	1.69±0.17##	1.42±0.12	1.37±0.10*	1.28±0.10**	1.18±0.10**

 

Table 3 Effects of CDPs on body mass of mice (n = 6)

 

Group	Time/Week
	0	1	2	3	4
Control	28.57±0.82	28.75±0.97	28.70±0.79	29.10±1.24	29.32±1.35
Model	24.53±1.25##	24.92±1.20##	24.35±1.39##	24.82±1.22##	24.73±1.56##
CDPs-L	25.75±0.69	24.77±0.91	24.83±0.99	25.10±0.83	25.42±0.97
CDPs-M	25.10±0.56	25.00±0.54	24.68±0.48	25.13±0.62	25.25±0.58
CDPs-H	25.38±0.28	25.43±0.49	25.52±0.62	25.73±0.50	26.08±0.34*
Dapa	25.13±1.02	25.52±0.98	25.97±1.00*	26.25±0.63*	26.63±0.78*

 

 

2.2 Effect of CDPs on renal function of C57BL/6J mice

As shown in Table 4, compared with the Control group, the BUN, Cre, and UA concentrations in serum and the 24 h UTP mass concentration in urine of mice in the Model group were significantly increased (P<0.01); compared with the Model group, CDPs-M and CDPs-H can significantly reduce the concentrations of BUN, Cre, and UA in serum and the 24-hour UTP mass concentration in urine (P＜0.05, P＜0.01). CDPs-L can significantly reduce the concentrations of BUN and UA in mouse serum. and 24 h UTP mass concentration in urine (P＜0.05, P＜0.01).

 

Table 4 Effects of CDPs on BUN, Cre, UA and 24 h UTP in DN mice (n = 6)

Group	BUN Concentration (mmol/L)	Cre Concentration (μmol/L)	24h UTP Content Concentration (mg/mL)	UA Concentration (μmmol/L)
Control	47.51±7.32	12.80±1.36	1.32±0.12	103.9±18.59
Model	157.97±30.57##	44.85±4.36##	2.38±0.37##	500.43±81.06##
CDPs-L	112.70±16.76*	40.72±3.62	1.37±0.12**	264.07±14.85**
CDPs-M	82.31±14.68**	35.37±6.21*	1.06±0.17**	247.62±11.22**
CDPs-H	64.58±8.64**	30.00±2.87**	0.65±0.17**	222.71±37.52**
Dapa	76.98±10.47**	28.82±3.72**	0.37±0.10**	129.87±17.69**

 

 

2.3 Effect of CDPs on inflammatory factors and LPS mass concentration in serum of C57BL/6J mice

 

As shown in Table 5, compared with the Control group, the serum levels of TNF-α, IL-1β, IL-6, TGF-β and LPS in the mice in the Model group were significantly increased (P<0.01); compared with the Model group, Each dose of CDPs can reduce the levels of TNF-α, IL-1β, IL-6, TGF-β and LPS in serum, CDPs-M
The effect of the CDPs-H group was significant (P＜0.05 or P＜0.01), and the effect of the CDPs-H group was significant (P＜0.01).

Table 5 Effects of CDPs on inflammatory factors and LPS in mice (n = 6)

 

Group	Mass Concentration (pg/mL)
	TNF-α	IL-1β	IL-6	TGF-β	LPS
Control	111.23±5.47	125.85±11.41	112.27±10.28	152.25±7.28	116.17±15.07
Model	167.90±16.91##	184.53±21.03##	195.57±31.88##	209.36±16.17##	173.51±15.72##
CDPs-L	144.57±10.15*	160.46±11.84*	169.10±20.32	189.03±8.02*	151.41±9.40*
CDPs-M	133.62±13.24**	154.30±17.39*	150.47±19.28*	171.25±14.35**	135.33±23.92**
CDPs-H	119.30±7.31**	140.54±16.11**	127.12±10.94**	169.47±10.57**	124.98±16.38**
Dapa	114.79±10.48**	135.17±14.37**	120.90±9.32**	159.11±10.92**	113.34±13.14**

 

 

2.4 Effect of CDPs on renal pathology of C57BL/6J mice

As shown in Figure 1, after H&E staining, it was found that the glomerular structure and morphology of the mice in the Control group were normal and clear, the basement membrane was smooth, the glomerular morphology was regular, the renal interstitium was normal, the renal tubules were neatly arranged, and there was no inflammatory cell infiltration. Compared with the Control group, the glomerular volume of the mice in the Model group was enlarged, the basement membrane was thickened, inflammatory cells were infiltrated around it, and the renal tubules were disordered and dilated; compared with the Model group, the CDPs administration groups had different degrees of improvement on the mouse kidneys. The glomerular volume of the mice in the CDPs-M and CDPs-H groups was significantly reduced, the basement membrane thickness was reduced, only a small amount of inflammatory cells were infiltrated, and the renal tubules recovered significantly and were tightly arranged. After 4 weeks of CDPs intervention, the above pathological conditions were all restored to varying degrees.

Masson staining showed that the renal tubules of the mice in the Control group were arranged normally and the glomeruli were in normal shape, no collagen fiber proliferation was observed, and tubular basement membranes were visible; a large amount of stained collagen was observed in the renal interstitial tissue of the mice in the Model group, and the interstitial fibrous tissue was bundled and reticularly proliferated; the glomerular volume of the mice in the CDPs-M group was reduced, and the symptoms of fibrous tissue proliferation were alleviated; the renal tubules of the mice in the CDPs-H group were arranged more regularly, and a small amount of collagen fiber proliferation was observed.

PAS staining results showed that the glomeruli of the mice in the Control group were normal in volume and intact in structure; the mice in the Model group had kidney damage, the glomerular volume was larger than normal and the shape was irregular, and mesangial matrix was deposited; compared with the Model group, the kidney damage of the mice in each drug-treated group was alleviated, and with the increase of the dose, the glomerular shape and size tended to be normal, and the degree of mesangial matrix deposition was reduced. The results suggest that CDPs can alleviate kidney tissue damage in DN mice.

 

Fig. 1 Effect of CDPs on renal histopathologic changes in DN mice as examined by H&E, Masson and PAS staining (× 400)