Chronic consumption of thermally processed palm oil or canola oil modified gut micro flora of rats

Mengcheng Run,Yirn Bu,Fngjie Wu,Shijie Zhng,Rulong Chen,N Li,Zhiguo Liu,?,Hulin Wng,?

aSchool of Biology and Pharmaceutical Engineering,Wuhan Polytechnic University,Wuhan,Hubei,430023,China

bHubei Provincial Key Laboratory for Applied Toxicology,Hubei Provincial Academy of Preventive Medicine,Wuhan 430079,China

cHubei Research Centre for Laboratory Animal,Wuhan 430079,China

Keywords:

Palm oil

Canola oil

Gut microbiota

Short-chain fatty acids

Thermally oxidized oils

ABSTRACT

Dietary oils have critical influences on human health,and thermally cooking or frying modify the components and nutritional functions of oils.Palm oil was the most widely used oil in food processing industry,but its health effects remain debatable.In the current study,we aimed to compare the effects of thermally oxidized palm oil and canola oil on gut microbiota.Palm oil or canola oil were heated at 180?C for 10h to prepare high-fat diets.Rats were fed high-fat diets for 3 months,and hematological properties,gut micro flora composition and intestinal gene expression were examined.The results indicated that heated canola oil consumption elevated plasma total cholesterol and LDL-c levels compared with unheated canola oil,but heated palm oil does not had these effects;and consumption of heated palm oil significantly elevated the relative abundance of Lactobacillucs and Roseburia in gut,compared with non-heated palm oil or two canola oil groups.Moreover,intestinal expression of IL-22 was increased in heated palm oil fed animal,though ZO-1 and GPR41 were reduced.In conclusion,heating process may enhance the effects of palm oil on proliferation of probiotics Lactobacillucs,and weaken the effects of canola oil on cholesterol transport and metabolism.

1.Introduction

Fat/lipid is one of macronutrients,provides energy,essential fatty acids(α-linolenic acid and linoleic acid)and fat-soluble vitamins.Recent clinical studies have demonstrated that high intake of fats may have little negative effects on health[1].In the latest“Dietary Guidelines for Americans”,the limitations on both cholesterol and total fat consumption were removed[2,3].The quality of dietary lipids is more important than the quantity,and we need more information for the heathy chosen of cooking fats[4].

Fats are widely used in cooking,particularly in frying.Frying is a process of heating food in oil at a high temperature over 150?C,decreasing the content of unsaturated fatty acids in oil and increasing the free fatty acids,polar materials,and polymeric compounds[5].Thereby,palm oil was believed to be suit for frying due to its high content of saturated fatty acids(SFAs),and it has been the world’s most widely used oil for food processing industry[6,7].However,dietary guidelines suggest to limit the consumption of saturated fats and replace to unsaturated fats,to reduce the risk of metabolic diseases such as cardiovascular disease and diabetes[2,3,8].Indeed,canola oil,the low erucic acid rapeseed oil rich in oleic acid,an omega-9 monounsaturated fatty acid(MUFA),presents benefits on cardiovascular disease and diabetes[9–12].However,unsaturated fatty acids contain carbon-carbon double bonds,susceptible to be oxidized in the high-temperature cooking methods,like frying[13].Whether substituting canola oil for palm oil in high-temperature frying is healthy,particularly for gut microbiota?In the current study,we aimed to explore the effects of thermally oxidized canola oil on gut microbiota compared with palm oil.

Gut microbiota plays a crucial role in glucose and lipids metabolism[14].Moreover,gut microbiota modify the host immune system and affect a serial of immuno-related diseases such as inflammatory bowel disease,non-alcoholic steatohepatitis,and even neurological diseases[15].The composition of gut microbiota were modified by both host and diets[16,17].Dietary fats have important influence on gut microbiota,many studies have demonstrated that high fat intake induced changes of gut microbiota at the phyla level and genus level,meanwhile the change of gut microbiota led to high-fat diet induced obesity and metabolic disorders[18,19].Different dietary oils have different effects on composition of gut microbiota:polyunsaturated lipids have been termed as prebiotics[20],and the effects of saturated or monounsaturated lipids on gut microbiota are more complicated. High-palm oil diet feeding elevated the relative abundance ofClostridialesspp.and inhibited the expanse ofBacteroidalesin a specific pathogen-free(SPF)mice model[21].Another study found that palm oil consumption stimulated the proliferation ofDesulfovibrionaceae,and had a reduced relative abundance ofRikenellaceae[22].However,the effects of thermally oxidized palm oil on gut microbiota remain unknown.A clinical study illustrated that canola oil-rich diet was associated with higher relative abundance ofStreptococcus,Tepidimicrobium,Robinsoniella,andTuricibacterin overweight people;g.Parabacteroides,Prevotella,Flexithrix,Fusibacter,f.Enterobacteriaceae,andp.Firmicuteswere correlated to canola oil in obese people[23].Zhou et al.[24]showed that non-heated canola oil feeding increased the relative abundance ofLactobacillus,and deep-fried canola oil inhibited the proliferation ofPrevotellain rats.In the current study,we compared the gut microbiota of rats fed fresh or heated canola oil and palm oil for three months,to reveal the influence of hightemperature heating on dietary fats.

2.Materials and methods

2.1.Animal models and treatment

Specific pathogen-free(SPF)Male Sprague Dawley rats(8weeks old,License no.42000600024039)were purchased from the Center for Disease Control of Hubei province(Wuhan,China)and were housed in a 12h day/night cycle with a room temperature of(20±3)?C.After 1 week of acclimatization,the rats were randomly distributed into the following five groups:Low-fat control group(CON),high unheated palm oil group(POC),high heated palm oil group(POH),high unheated canola oil group(COC),high heated canola oil group(COH)for 3 months,and 6 rats per group.The high-fat diets were made by adding a specific oil at a mass ratio of 23% weight instead of corn starch(10%)and maltodextrin(13%).The formula of diets matched the AIN-93 requirement.The heated oils were heated at a high temperature of 180?C for 10h.The animals were given water and dietad libitum.This animal study was conducted according to the Guidelines for the Care and Use of Experimental Animals,and the protocol was approved by Laboratory Animal Ethics Committee of Wuhan Polytechnic University(ID number:2017101501).Every month,we collected their droppings,kept at?80?C for following experiments.

2.2.Oil fatty acids analysis by GC–MS

Methyl esterification of different kinds of oil was referred to our previous literatures[25,26].Methylated oil was subjected to gas chromatography-mass spectroscopy(GC–MS).GC–MS equipment consists of a polor HP-88 capillary column(100m×0.25mm id.0.25μm film thickness,Agilent,CA).Helium was used as carrier gas and the flow rate of mobile phase was set at 1mL/min.The oven temperature of the instrument was raised from 100?C to 260?C at 10?C/min and injection volume was set at 2μL.For GC–MS detection,an electron ionization energy system with ionization energy of 70eV was used.The total running time for a sample is 45min.Samples that dissolved in n-hexane and methanol were run fully at a range of 10–850m/z,and the results were compared using Agilent’s MSD ChemStation(E.02.00.493,Agilent Technologies,Inc.,USA),The name,molecular weight,molecular formula,and structure of the component of test materials were determined,while the relative percentage of each component was calculated by comparing its average peak area to the total areas.

2.3.Histological studies and biochemical measurements

At the end of 14th week of feeding,all rats were sacrificed by way of CO2suffocation,weighed,Blood samples were collected,and distributed into a heparinized tube(10mL)and centrifuged at 1000×gfor 15min at 4?C,and plasma was collected and stored at?80?Cuntil analysis.Concurrently,the intestine and the liver were rapidly removed,rinsed with 0.9% sodium chloride solution,with intestine cutting into 3 parts(small intestine,large intestine and cecum).Then kept at?80?C or fixed in 10% neutral buffered formalin.The levels of total cholesterol(TG),triacylglycerol(TC)and low density lipoprotein cholesterol(LDL-c)were analyzed using an automated biochemical analyzer(Nanjing Jiancheng Bioengineering Institute,China).Fixed tissue were processed for HE staining for following-up histopathological diagbosis.

2.4.Fecal short-chain fatty acids(SCFAs)analysis by GC–MS

Quantification analysis of fecal SCFAs was the same as the described method[27]and was performed using an Agilent 7890A gas chromatography coupled with an Agilent 5975C mass spectrometric detector(Agilent Technologies,USA).For samples of feces,fecal water was prepared by homogenizing feces in 0.005mol/L aqueous NaOH followed by centrifuging at 13200×gat 4?C for 20min.The supernatant fecal water was derivatization with PrOH/pyridinemixture solvent(3:2,V/V)and propyl chloroformate(PCF).After derivatization,the derivatives were extracted by a two-step extraction with hexane.The concentrations of the SCFAs(acetic acid,propionic acid,butyric acid,isobutyric acid,and nvaleric acid)were performed with a polar HP-5 capillary column(30m×0.25mm id.0.25μm film thickness,Agilent,CA).Helium was used as a carrier gas at a constant flow rate of 1mL/min.The initial oven temperature was held at 60?C for 5min,ramped to 110?C at a rate of 10?C/min,then ramped to 250?C at a rate of 35?C/min and finally held at this temperature for 1min.The temperature of the frontinlet,transfer line,and electron impact(EI)ion source was set as 280,250,and 230?C,respectively.Data handing was performed with an Agilent’s MSD ChemStation(E.02.00.493,Agilent Technologies,Inc.,USA).

2.5.Fecal DNA extraction,16S rDNA gene sequencing

Total genome DNA from samples was extracted using CTAB/SDS method.The hypervariable regions V3 and V4 of the 16S rDNA genes were PCR amplified with the primer 338F-806R,F:5＇-ACTCCTACGGGAGGCAGCA-3＇and R:5＇-GGACTACCGGGGTWTCTAAT-3＇.16S rDNA genes were amplified using the specific primer with the barcode.All PCR reactions were carried out in 30μL reactions with 15μL of Phusion?High-Fidelity PCR Master Mix(New England Biolabs);0.2μmol/L of forward and reverse primers,and about 10ng template DNA.Thermal cycling consisted of initial denaturation at 98?C for 1min,followed by 30 cycles of denaturation at 98?C for 10s,annealing at 50?C for 30s,and elongation at 72?C for 30s.Finally,70?C for 5min.PCR products was mixed in equidensity ratios.Then,mixture PCR products was purified with GeneJET Gel Extraction Kit(Thermo Scientific).Sequencing libraries were generated using NEB Next?UltraTMDNA Library Prep Kit for Illumina(NEB,USA)following manufacturer’s recommendations and index codes were added.The library quality was assessed on the Qubit?2.0 Fluorometer(Thermo Scientific)and Agilent Bioanalyzer 2100 system.At last,the library was sequenced on an Illumina HiSeq platform and 250 bp paired-end reads were generated.

2.6.Quantification of intestinal bacteria by quantitative real-time PCR

Fecal DNA was obtained using the TIANamp Stool DNA Kit(TIANGEN).The quantification was performed using 16S universal or bacterial specific rDNA gene primers and quantitative RT-PCR.Differences(ΔCT)between 16S CT values and specific bacterial groups were used to obtain normalized levels(2?ΔΔCT).The relative abundance of each bacterial group was obtained after normalization with the control groups.Primer sets used for q-PCR are listed in Table 1.

2.7.RNA extraction and quantitative real-time PCR

The extraction of total RNA from colon samples was carried out using Trizol(Takara,Japan)following the manufacturer’s instructions.cDNA was obtained using a reverse transcription kit(Takara,Japan)using the T100 gene amplification instrument(Bio-rad,USA).Real-time quantitative PCR(qPCR)was performed with iTaqUniversal SYBR Green SuperMix,plus each oligonucleotide,in a 96-well plate format.The amplification program consisted of:1)pre-incubation at 95?C for 2min;2)40 cycles of denaturation at 95?C for 15s and annealing at 60?C for 30s on the CFX96(Bio-rad,USA).Melting curve profiles were produced to ensure product specificity.Expression levels were normalized to β-actin following the ΔΔCt algorithm.Primer sets used for q-PCR are listed in Table 1.

Table 1Primer sequences.

Fig.1.Effects of 14 weeks feeding of control diet(CON),non-heated palm oil diet(POC),heated palm oil diet(POH),non-heated canola oil diet(COC),heated canola oil diet(COH)on body weight(A),body weight change(B)and liver histology(hematoxylin and eosin(H&E)staining,magnification 100×,200×)(C).Data are expressed as mean±SD(n=6 per group).

Table 2Fatty acids composition of non-heated or heated oils.

2.8.Statistical analysis

The data are expressed as mean ± SD. Principal coordinates analysis was performed with the cmdscale command(stats package for R).All statistical analyses were performed using R v.3.5,and if necessary,Pvalues were corrected using the Benjamini-Hochberg correction.Data were statistically analyzed with Prism 7.0(Graph-Pad,La Jolla,CA,USA).Statistical significance was determined by 1-or 2-way repeated-measures ANOVA with the Mann-WhitneyUtest.ThePvalues were calculated,and null hypotheses were rejected whenP＜0.05.

3.Results

3.1.High-fat diets consumption has not induced obesity or hepatic steatosis in rats

The main fatty acids components of non-heated palm oil(POC),heated palm oil(POH),non-heated canola oil(COC),or heated canola oil(COH)were listed in Table 2.We found that palm oil was rich in SFAs and MUFAs,but lack of polyunsaturated fatty acids(PUFAs),canola oil rich in monounsaturated oleic acid,and contained less than 1% of erucic acid.Moreover,heating process slightly reduced the content of PUFAs and increased SFAs in both palm oil and canola oil.

After three months feeding,no obesity was observed in all four high-fat diet groups(POC,POH,COC,COH)compared with the CON group(Fig.1A,1B).Moreover,hepatic histological staining illustrated that high-fat diets did not induced ectopic lipid accumulation in liver(Fig.1C).

3.2.Oxidized canola oil consumption inducedhypercholesterolemia and slight insulin resistance

In the current study,none of the four high-fat diet groups presented typical hypertriglyceridemia after three months feeding(Fig.2A).It is always concerned that saturated fat intake is inclined to raise the LDL-c level and induce hypercholesterolemia,thereby elevating the risk of cardiovascular diseases.However,in the current study,the TC levels of animals in both non-heated and heated palm oil groups were not higher than low fat CON group,and interestingly,consumption of high-temperature heated canola oil led to a significantly increased TC level compared with both CON group and non-heated COC group(Fig.2B).Concordantly,the LDL-c level of COH group was significantly higher than CON group,and other three high-fat groups also had a slightly increased LDL-c level(Fig.2C).

Fig.2.Effects of three months feeding of heated oils on plasma total cholesterol(TG,A);triacylglycerol(TC,B);low density lipoprotein cholesterol(LDL-c,C);fasting blood glucose(GLU)levels(D);and area under the curve(AUC)of glucose tolerance tests(OGTT,F).Data are expressed as mean±SD(n=6 per group).*,P＜0.05;**,P＜ 0.01;***,P＜0.001;respectively.

Fig.3.The effects of high-fat diets on gut microbiota.Heatmap of gut microbiota from feces of rats fed one month of high-fat diets at phylum level(A)and genus level(B).Relative abundance of specific bacteria in feces of animals fed three months of high-fat diets:Butyrivibrio(C);Lactobacillucs(D);Roseburia(E);and Prevotella(F).*,P＜0.05;**,P＜0.01;***,P＜0.001;respectively.

Excessive lipid accumulation,particular the accumulation of intermediate products like diglyceride may induce peripheral insulin resistance.However,in the current study,we found that fasting glucose level of rats in high-fat diet feeding groups were only slightly but not significantly higher than CON group(Fig.2D).The OGTT showed similar results,high-fat diet consumption had not induced glucose intolerance(Fig.2E).

3.3.High-fat diets consumption modified gut microbiota in rats

After one month of feeding,feces of animals in each group were equivalently mixed for DNA extraction and 16S rDNA sequencing.Identified species of microorganisms in each group were more than 800,except heated palm oil group.Index of alpha diversity,such as Shannon,Simpson,chao1 and ACE indicated no difference betweeneach two groups(Table 3),suggests that high-fat diets have no effects on richness of microbiome.

Table 3Abundance and diversity indexes of microbiota of five groups.

Fig.4.Effects of thermally oxidized oils on fecal short-chain fatty acids:Acetate(A);Propronate(B);and butyrate(C).Data are expressed as mean±SD(n=6 per group).

At phylum level,ten major bacteria phyla were detected in gut microbiomes of animals fed high-fat diets,andBacteroidesandFirmicuteswere the dominant phyla.The relative abundance ofFirmicutesin both palm oil fed groups and heated canola oil fed group were higher than control group,but that in non-heated canola oil group was lower than both control and heated canola oil groups,implies high temperature processing of canola oil may change its effects on gut microbes(Fig.3A).The ratio ofFirmicutestoBacteroidetesin CON,POC,POH,COC and COH were 1.08,1.53,2.21,0.70 and 1.61,respectively.Moreover,the relative abundance ofTenericutesin COH group was lower than other groups.

Furtherly,community heatmap analysis at genus level illustrated that the structure of gut microbiota has been modified after one month of high-fat diet consumption.The relative abundance ofRoseburiawas increased in palm oil feeding groups,particularly non-heated palm oil group,and heated palm oil group had the highestLactobacilluslevel;relative abundance ofButyrivibrioandRuminococcaceae_UCG-005 was higher in heated canola oil group than other groups; and non-heated canola oil group has the highestRuminococcaceae_NK4A214group level(Fig.3B).

To verify the effects of high-fat diets on these bacteria, after three months feeding,feces sample of each rats were collected for DNA extraction and PCR amplification of 16S rDNA test.Data showed consistent results of relative abundance change ofButyrivibrioandLactobacilluswith one month feeding(Fig.3C,D),suggested that the effects of oils on gut microbiota were persistent.The relative abundance ofRoseburiawas found higher in heated palm oil group than non-heated group,but lower than control group(Fig.3E).The relative abundance ofPrevotellahad no difference between each group(Fig.3F).

3.4.High-fat diets feeding had little effects on SCFA in feces

After three months feeding of high-fat diets,feces sample were collected for SCFA measurement,and results indicated that highfat diets feeding had little effects on fecal acetate level(Fig.4A),propionate and butyrate level were slightly lower in four high-fat groups compared with control group with no significance, thoughPvalue between POH and CON group was 0.0563 for butyrate(Fig.4B,4C).

3.5.Intestinal expression of particular genes was modified by high-fat diets feeding

To examine the effects of thermally oxidized oils on intestinal functions,expression levels ofGPR41,ZO-1,IL-1β andIL-22in colon tissues were measured using RT-qPCR.Consistent with SCFAs results,the expression level ofGPR41in POH group was significantly lower than CON group(Fig.5B).High-fat diets feeding reduced the expression of tight junction geneZO-1,except nonheated palm oilgroup(Fig.5C),though no remarkable changes were observed on intestinal histology(Fig.5A).We then checked the colon structure of rats,and histological staining of colon illustrated that high-fat diets induced visible change of intestinal structure.Expression of two intestinal cytokinesIL-1β andIL-22were measured,the expression ofIL-1β was inhibited by high-fat diets consumption(Fig.5D), and interestingly, heated palm oil consumption boosted the expression ofIL-22in intestine(Fig.5E).

4.Discussion

Dietary guidelines recommended to substitute unsaturated vegetable oils for palm oil or other saturated fats[2].However,the effects of palm oil on health was still debatable.Excessive consumption of palm oil was considered to cause health problems,such as raising the morbidity and mortality of cardiovascular disease[28,29].On the other hand,many studies indicated that intake of palm oil elevated the level of both “bad”cholesterol,LDL-c and“good”cholesterol,HDL-c,and no strong evidences were found to link palm oil consumption and cardiovascular disease[30–32].Our previous study had revealed animal-source saturated fats may cause chronic liver damage,and monounsaturated fatty acids enriched lipids had protective effects against liver injury[33,34].In the current study,comparison of palm oil with canola oil showed no difference on body weight, bodyweight change or liver histology(Fig.1A–1C).Moreover,three-months consumption of unheated palm oil or canola oil presented no effects on TC,and levels of LDL-c were increased in both POC and COC group with no significance(Fig.2B,2C).After 10h heating at 180?C,heated canola oil significantly elevated both TC and LDL-c levels compared with the low-fat control diet(Fig.2B,2C).Zhang et al.[24]have reported the effects of deep-fried canola oil on TC or LDL-c on their study,and another study found that intake of dietary oxidized frying soybean oil in guinea pigs increased the plasma TC level,but most of animal studies indicated that oxidized soybean oil or sun flower oil had no effects or even reduced TC[35,36].Some clinical studies have found that consumption of canola oil can reduce the TC or LDL-c level compared with saturated fat[37],however,some other studies showed that oleic acid rich diets may increase the LDL-c level[38],and a study from Peter J.H.Jones and colleagues[39]indicated the high canola oil diet induced LDL-c level elevation was due to enrichment of cholesteryl oleate,but not LDL-proteoglycan binding,suggests the canola oil associated LDL may not be associated with increased risk of atherosclerosis.

Long time high-fat diets consumption were always associated with insulin resistance[40].We observed that intake of high-fat diets led to slightly increased fasting glucose levels or higher curve of OGTT,but no significance was found(Fig.2D,2E).Li et al.[41]also observed no effects of deep-fried oxidized palm oil on fasting glucose level or glucose tolerance in mice.A longer period feeding study may be needed to reveal the effects of oxidized oils on insulin action and glucose metabolism.

Gut microbiota was believed to play a crucial role in highfat diet induced obesity and metabolic disorder[21,22].In the present study,we found that three months high-fat diets feeding had no effects on gut diversity,but modified the structure of gut microbiota.Furthermore,high-temperature processing of oils had remarkably influence on gut microbes.Intake of oxidized oils increased the relative abundance of Firmicutes at phylum level,thereby Firmicutes/Bacteroides ratio was elevated(Fig.3A).Firmicutes and Bacteroidetes are the two most predominant phyla in intestine,and higher abundance of Firmicutes was suggested to be linked to higher energy harvest capability from fermentation and SCFAs production[42].However,our SCFAs analysis had not found any difference of acetate or propionate level between each groups,and the fecal butyrate level was lower in oxidized palm oil fed group,which was consistent with the reduced relative abundance of butyric acid produced germButyrivibrio(Figs.3C,4 A–4C).Butyric acid is important for host health,it can activate the intestinal fatty acid receptor GRP41 and induce downstream signals[43].Not surprised,the intestinal expression level of GRP41 was reduced in POH group(Fig.5B).These findings suggested that intake of oxidized palm oil may reduce the capability of butyric acid production in gut.The decreased butyrate level in POH group may furtherly weaken the gut barrier,as the expression of tight junction protein ZO-1 was inhibited compared with CON group(Fig.5C).

On the other hand,oxidized palm oil consumption stimulated the expansion ofLactobacillus(Fig.3E).It was a well-known probiotics and produced lactic acid,which can lower luminal pH in colon and help to limit the proliferation of pathogens[44].Oxidized palm oil may influence the fermentation utilization mode of intestinal bacteria on proteins,thus promoting the proliferation oflactobacillus,but the mechanism remained to be further studied.Moreover,Lactobacillicatalyze the conversion of tryptophan to indole derivatives,which can promote the production of IL-22[45].IL-22 was produced by innate lymphoid cells group 3(ILC3)and play crucial roles in intestinal bacterial infection controlling,intestine repairing,and recent studies suggest that IL-22 may take part in the regulation of glucose and lipid metabolism[46,47].Coincident with the increased abundance ofLactobacillus,the expression of IL-22 was elevated in oxidized palm oil fed animals(Fig.5E).

In four high-fat diet groups,Roseburiawas only presented in POH group(Fig.3D).Roseburiais an intestinal commensal bacterium which was suggested to have benefits on host health,as protection host against atherosclerosis[48].The effects of oxidized palm oil to keepRoseburiaalive may contribute to reduce the risk of saturated fat induced cardiovascular disease.

Gut microbiota modulate host immune system,and closely associated with peripheral inflammation[49].In the current study,we found that high-fat diets consumption reduced the intestinal expression of pro-inflammatory cytokine IL-1β(Fig.5D),implies that high-fat consumption may reduce the activation of intestinal macrophages and had not induced inflammation in gut.

In conclusion,our study found that high-temperature heating has complicated effects on the nutritional value of dietary fats,thermally oxidized canola oil consumption affected the transport and metabolism of cholesterol,induced hypercholesterolemia and elevated LDL-c level;as well as heated palm oil stimulated the proliferation ofLactobacillusandRoseburia,two probiotics-type germs which were believed have benefits on health,and induced the expression of IL-22 in gut.The chosen of cooking oils in food industry should be full considered of their influence on health,including the gut microbiota.

Declaration of Competing Interest

There are no conflicts of interest to declare.

Acknowledgements

This study was supported by Hubei Provincial Natural Science Foundation of China(Grant No.2017CFB275),National Natural Science Foundation of China(Grant Nos.31271855 and 81402669),and the Fundamental Research Funds for the Wuhan Polytechnic University(2019J04).