Study of the effect of Jian-Pi-Xiao-Shi formula on infantile rats with anorexia

Yu-Jing Shi,Jia Liang,Wen-Yi Zhu,Hong Meng＊

1Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;2Graduate School of Chinese Academy of Traditional Chinese Medicine,Beijing 100700,China;3Beijing Technology and Business University,Beijing 100048,China.

Keywords:Jian-Pi-Xiao-Shi formula,Infantile anorexia,Orexigenic hormone,Digestive function

Background

Anorexia is a common problem in pediatrics,with trends increasing in recent years.Its main clinical presentations include a reduced appetite,decreased food intake,and delayed weight gain over a long period of time [1].The main causes of pediatric anorexia are irrational feeding,improper dietary composition,and fatty and greasy diets[1–4].Pediatric anorexia can cause malnutrition,trace element deficiency,and weakened immunity,which severely affect pediatric growth and development as well as the overall mental and physical health[1].At present,most experimental studies use a rat model of pediatric anorexia,developed by feeding juvenile rats a high protein,high fat feed (hereinafter referred to as special feed)for a 4-week period.The levels of many minerals and vitamins are low in special feed,which fits the natural etiology of pediatric anorexia.Special feed intake can result in a significant decrease in food intake,delayed weight gain [2–6],decreased serum iron and copper levels [2],decreased pepsin activity due to long-term feeding[3],and changes in peripheral blood gastrointestinal hormone secretion.Moreover,studies on these rat models have found increased cholecystokinin octapeptide (CCK-8) [4,5]and decreased β-endorphin (β-EP) [4,5],motilin (MTL),and gastrin(GAS)levels[6].

Chinese patent drug (CPD) Jian-Pi-Xiao-Shi formula (JPXSF) is made from plant material used in traditional Chinese medicines,such as Shanzha(Crataegi fructus),Shanyao (Dioscoreae rhizoma),Yiyiren (Coicis semen),Fuling (Poria),Qianshi(Euryales semen),Wumei (Mume fructus),and Dazao(Jujubae fructus) and foods such as Jinzhengu(Flammulina velutipes),Xianggu (Lentinula edodes),and Yiner (Tremella fuciformis),which have gastrointestinal nourishing functions and are rich in elemental nutrients.CPD JPXSF has“Spleen-Invigorating”, “Digestion-Aiding”,and“Qi-Regulating”effects.In this study,a juvenile rat model of anorexia was constructed by using a 28-day special feed diet.During model construction,CPD JPXSF was administered orally,and its effects were evaluated by measuring animal weight,gastrointestinal function,and expression of gastrointestinal hormones.

Materials

Experimental animals

Four-week-old Sprague Dawley male and female rats(specific-pathogen-free grade) weighing 50–70 g were purchased from Beijing HFK Bioscience Co.Ltd.,animal qualification certificate no:11401300089682,usage license:SYXK (Beijing) 2015-0041.The experiments were conducted after obtaining approval from the ethical committee (Animal ethics approval no.:2019D013).

Drugs and reagents

CPD JPXSF granules are made fromCrataegi fructus,Dioscoreae rhizoma,Coicis semen.Poria,Euryales semen,Mume fructus,Jujubae fructus,Flammulina velutipes,Lentinula edodes,andTremella fuciformis.Aqueous extracts of these substances were concentrated followed by spray drying and sieving.The granules were manufactured by Infinitus (China)Company Ltd.(batch number:19C14HAS01EA).In clinical practice,the dose of CPD JPXSF granules used in 5–7-year-old children is 4.5 g/20 kg/d.In this experiment,this was converted to an equivalent dose(1.20 g/kg/d),which was used as a medium dose in the rats used in our study.Two additional doses were used:a high dose (2.40 g/kg/d),which represented twice the equivalent dose,as well as a low dose (0.60 g/kg/d),representing half an equivalent dose.The special feed(fish floss,milk powder,corn flour,soy flour,sugar,fresh eggs,and lard in a ratio of 1:1:1:2:1:1.8:2)was manufactured by Beijing HFK Bioscience Co.Ltd.(Batch no.:1103221800002944).Other materials used include a pepsin assay kit (A080-1,Nanjing Jiancheng Bioengineering Institute),(CSB-E12743r,CUSABIO),a rat MTL enzyme-linked immunosorbent assay(ELISA) kit (CSB-E08208r,CUSABIO),a rat CCK-8 ELISA kit (CSB-EQ027822RA,CUSABIO),and a rat beta endorphin ELISA kit(CSB-E08206r,CUSABIO).

Instruments

A MULTISKAN MK3 fully automatic multi-mode microplate reader(Thermo Scientific Inc,USA),and a 5810R high-speed refrigerated centrifuge (Eppendorf AG,Germany)were used.

Methods

Grouping and dosing

Sprague Dawley rats were randomized into the following five groups by weight:normal group,model group,and high,medium,and low-dose CPD JPXSF groups.Each group contained 6 male and 6 female rats,with 3 rats per cage.With the exception of the normal group,rats were given ad libitum access to special feed for 28 consecutive days to induce weight loss and decreased weight gain rate,thus creating a rat model of anorexia.Rats in the normal group were given ad libitum access to normal growth feed.With the exception of the normal group and model group,other groups were given the corresponding investigational drug (CPD JPXSF) or control feed by gavage (1 mL/100 g,qd,for 28 consecutive days).Rats in the normal and model groups were given distilled water under the same conditions.

Weight and food intake

Rats were weighed once every seven days,and weight gain and weight gain rate were calculated.After weighing on every seventh day,the quantity of special feed in each feed was changed to an equivalent amount of normal growth feed and the weight of the remaining feed in each cage was measured after 24 hours to calculate the 24 h food intake.Weight gain in grams was calculated with the following formula:weight gain(g)=rat weight at each week after model construction(g)?rat weight before model construction(g).

Biomarker measurement

After feed weight was measured on day 29,rats were fasted for 12 hours and blood was then collected from the abdominal aorta.Samples were centrifuged at 4,000 rpm for 15 minutes to isolate serum.Serum was stored at ?20 ℃and an ELISA was used to measure β-EP,CCK-8,GAS,and MTL expression levels.Rats were autopsied and both ends of the stomach were ligated.Three milliliters of physiological saline was injected into the stomach and the gastric lavage was collected and centrifuged at 4,000 rpm for 10 minutes.The supernatant was collected,and colorimetry was used to measure pepsin expression levels.

Statistical methods

Statistical analysis was performed on all data using GraphPad Prism 7.0.Data were expressed as,and a one-way analysis of variance was used for inter-group comparisons.

Results

Effects on weight in juvenile anorexic rats

Rat weight was measured before model construction and during weeks 1–4 after model construction.There were no significant differences in weight between the various groups before model construction (P＞0.05).Weight gain in the normal group was 48.77 ± 6.39 g after week 1,85.07 ± 16.99 g after week 2,127.21 ±32.80 g after week 3,and 167.99 ± 42.82 g after week 4.At various time points between weeks 1 and 4,rat weights in the model group decreased significantly compared with the normal group (P ＜0.05,P ＜0.01),and weight gain was also significantly decreased compared with the normal group (P ＜0.05,P ＜0.01).The weights for the model group were 38.63 ± 7.14 g,51.54±5.28 g,101.20±21.28 g,and 131.68±36.83 g,for weeks 1–4,respectively.On weeks 1 and 2 of gavage with CPD JPXSF,the weights of the various dosing groups were significantly increased compared with the model group (P ＜0.05,P ＜0.01).Weight gain was also significantly higher in the CPD JPXSF group when compared to the model group (P ＜0.01),with the low-dose group showing the highest weight gain (55.45 ± 6.82 g and 104.85 ± 15.97 g after weeks 1 and 2,respectively).On week 3 of gavage with CPD JPXSF,the weights of the various dose groups significantly increased compared with the model group(P ＜0.01) and weight gain was also significantly higher than the model group (P＜ 0.01).The medium-dose group showed the highest weight gain for that week,with a value of 145.53 ± 42.90 g.On week 4 of gavage with CPD JPXSF,the weights of the various dose groups significantly increased compared with the model group(P ＜0.05)and weight gain in the high-and medium-dose groups were significantly higher than the model group (P＜ 0.05).The medium-dose group showed the highest weight gain,with a value of 171.11 ± 49.93 g.In weeks 1–4 of gavage with CPD JPXSF,there were no significant differences in body weight and weight gain among the high-,medium-,and low-dose groups (P＞ 0.05)(Figure 1,Table 1).

Effects on the digestive function in juvenile anorexic rats

Food intake during weeks 1–4 after model construction and pepsin expression in gastric juice at week 4 after model construction were observed in rats.Food intake decreased in the model group from week 1 of feeding with the special feed (Table 2).Food intake in the model group was significantly lower across all four weeks when compared with that observed in the normal group (P＜0.05).Food intake during weeks 1–3 for the various dosage groups of CPD JPXSF was significantly increased compared with the model group(P＜0.05,P＜0.01).At week 4 of administration,food intake in the high-dose group was significantly higher compared with the model group (P＜0.05).During weeks 1–4 of gavage with CPD JPXSF,there were no significant differences in food intake between the high-,medium-,and low-dose groups(P＞0.05).

Figure 1 Effects of Jian-Pi-Xiao-Shi formula on body weight in juvenile anorexic rats (,n=12). #P ＜0.05,##P ＜0.01 compared with normal group;*P ＜0.05,**P ＜0.01 compared with model group.

Table 1 Effects of Jian-Pi-Xiao-Shi formula on body weight in juvenile anorexic rats(,n=12)

Table 1 Effects of Jian-Pi-Xiao-Shi formula on body weight in juvenile anorexic rats(,n=12)

#P ＜0.05,##P ＜0.01 compared with normal group;*P ＜0.05,**P ＜0.01 compared with model group.

Table 2 Effects of Jian-Pi-Xiao-Shi formula on food intake in juvenile anorexic rats(,n=12)

Table 2 Effects of Jian-Pi-Xiao-Shi formula on food intake in juvenile anorexic rats(,n=12)

#P ＜0.05,##P ＜0.01 compared with normal group;*P ＜0.05,**P ＜0.01 compared with model group

Pepsin content in the model group was significantly lower than in the normal group after 4 weeks of model construction with the special feed (P＜0.01;Table 3,Figure 2).After four weeks of CPD JPXSF consumption,pepsin content in the high-dose group was significantly higher than in the model group (P＜0.05);the medium-and low-dose groups showed a nonsignificant increase in pepsin content compared with the model group (P＞0.05).At week 4 of gavage with CPD JPXSF,there were no significant differences in food intake among the high-,medium-,and low-dose groups(P＞0.05).

Effects on appetite hormones in juvenile anorexic rats

The expression levels of appetite-related hormones(β-EP,CCK-8,GAS,and MTL) in the blood of rats after four weeks of model construction were measured.In the model group (Table 4),β-EP and MTL expression levels were significantly lower than in the normal group (P＜ 0.01);β-EP expression in the various CPD JPXSF dose groups was significantly increased compared with the model group (P＜0.01,Figure 3A),and MTL expression in the high-dose group was significantly higher than in the model group(P＜ 0.05,Figure 3D).CCK-8 was significantly increased in the model group compared with the normal group (P＜0.01)and significantly decreased in the high-dose group compared with the model group(P＜ 0.01,Figure 3B).There were no significant changes in GAS expression between any groups (P＞0.05,Figure 3C),and no significant differences in β-EP,CCK-8,GAS,and MTL expression between the high-,medium-,and low-dose groups(P＞0.05).

Discussion

In traditional Chinese medicine,pediatric anorexia is a disease which encompasses the absence of cravings,poor appetite,and a dislike for eating,usually over 1–2 months [7].Pediatric anorexia differs from poor appetite seen in other acute and chronic diseases.This disease is mostly seen in children between 1 and 6 years old,and incidence is higher in urban areas than in rural areas.Western medicine considers anorexia to be an abnormal eating behavior that may or may not be accompanied with abnormal gastrointestinal function.The primary clinical characteristics are lack of interest in eating/refusal to eat and insufficient dietary intake,and is accompanied by insufficient growth or malnutrition[8–10].

Currently,modern medicine [11]regards insufficientgastrointestinal motility and decreased digestive absorption as the pathogenesis of pediatric anorexia.Deficiency of trace elements such as zinc,selenium,and copper,and vitamin A can also lead to pediatric anorexia.Gut dysbiosis is also one of the etiologies of pediatric anorexia.Furthermore,the feeding center of the central nervous system and the dysregulation of gastrointestinal hormones are other popular research avenues explored in modern studies to better understand the pathogenesis of anorexia.Traditional Chinese medicine believes that children have“Immature Yin and Yang”,“Delicate and Tender Visceral Organs”,“Insufficient Qi Synthesis”,and a characteristic of“Three Deficiencies and Four Excesses”.“Spleen Deficiency”is the most common traditional Chinese medicine diagnosis,and factors that affect“Spleen”and“Stomach”function can lead to anorexia.Common causes include milk stagnation,innate deficiency,“Spleen Damage”,and mental depression.Studies have shown that food stagnation and improper feeding (53.1%) are main causes of pediatric anorexia[12].

Table 3 Effects of Jian-Pi-Xiao-Shi formula on pepsin in juvenile anorexic rats(,n=12)

Table 3 Effects of Jian-Pi-Xiao-Shi formula on pepsin in juvenile anorexic rats(,n=12)

##P ＜0.01 compared with normal group;**P ＜0.01 compared with model group.

Figure 2 Effects of Jian-Pi-Xiao-Shi formula on pepsin in juvenile anorexic rats (,n=12). Inter-group comparison:*P ＜0.01,**P ＜0.05.JPXS,Jian-Pi-Xiao-Shi formula.

Figure 3 Effects of Jian-Pi-Xiao-Shi formula on appetite hormones in juvenile anorexic rats(ˉx ± s,n=12).Intergroup comparison:*P ＜0.01,**P ＜0.05.JPXS,Jian-Pi-Xiao-Shi formula.

Table 4 Effects of Jian-Pi-Xiao-Shi formula on appetite hormones in juvenile anorexic rats(,n=12)

Table 4 Effects of Jian-Pi-Xiao-Shi formula on appetite hormones in juvenile anorexic rats(,n=12)

#P ＜0.05,##P ＜0.01 compared with normal group;*P ＜0.05,**P ＜0.01.CCK-8,cholecystokinin octapeptide;β-EP,β-endorphin;MTL,motilin;GAS,gastrin.

CPD JPXSF is made from traditional Chinese medicines which includeCrataegi fructus,Dioscoreae rhizoma,Coicis semen.Poria,Euryales semen,Mume fructus,Jujubae fructus,and foods such asFlammulina velutipes,Lentinula edodes,andTremella fuciformis,and has “Spleen-Invigorating”,“Digestion-Aiding”,and“Qi-Regulating”effects.Crataegifructushas “SSpleen-Invigorating”,“Appetite-Stimulating”,and“Digestion-Promoting”effects,and is especially used as a drug for digesting greasy food.Dioscoreae rhizomahas“Spleen and Stomach Nourishing” effects and“Lung-Strengthening”effects.Coicis semencan“Nourish the Spleen and Stop Diarrhea,Remove Dampness,and has Diuretic”effects.Poriacan“Nourish the Heart and Spleen,Remove Dampness,has Diuretic effects,can Strengthen Body Resistance,and Remove Toxins”.Euryales semenhas“Kidney Nourishing,Resistance Strengthening,Spleen Supplementing,and Dampness Removing”effects.Mume fructushas a“Sour Taste”,is“Cool”and“Astringent”,can“Nourish Yin”,“Decrease Astringent Heat”,and“Nourish the Stomach”.Jujubae fructushas“Spleen-Invigorating,Qi-Replenishing,Spleen-,Stomach-,and Blood-Nourishing,and Mentally Calming”effects.Flammulina velutipes,Lentinula edodes,andTremella fuciformisare traditional edible fungi that contain polysaccharides,glycoproteins,proteoglycan,flammulins,and other bioactive substances [13].These substances have antineoplastic and immune-regulating properties,offer hepatocyte protection,and anti-fatigue effects [14–17].Flammulina velutipes is rich in proteins,amino acids,and many minerals.Frequent consumption can prevent and treat liver diseases and gastrointestinal ulcers,and promote both physical and mental development in children[15].

This study demonstrated that juvenile rats fed with special feed for 28 days started to show delayed weight gain and decreased food intake as early as week 1,and the juvenile anorexic rat model was successfully constructed.CPD JPXSF can significantly increase body weight in anorexic juvenile rats,of which weight gain rate was the highest in the 0.60 g/kg/d dose group in weeks 1 and 2 and in the 1.20 g/kg/d dose group in weeks 3 and 4.The 3 doses (2.40 g/kg/d,1.20 g/kg/d,and 0.60 g/kg/d) of CPD JPXSF can significantly increase food intake in anorexic rats in the first three weeks,and the 2.40 g/kg/d dose can continue to significantly increase food intake in week 4.

Pepsin can hydrolyze proteins in food and promote absorption.Hence,decreased pepsin expression reflects a decline in gastrointestinal function [18,19].After juvenile rats were fed with special feed for 4 weeks,pepsin level in rat gastric juice decreased.However,administration of 2.40 g/kg/d CPD JPXSF for 4 weeks significantly increased pepsin activity in rat gastric juice.

Many appetite-related polypeptide hormones play important roles in the regulation of feeding.For example,CCK-8 and β-EP are a pair of antagonistic hormones in the hypothalamus that jointly participate in the physiological regulation of feeding,and play important roles in pathological decrease in food intake[20,21].β-EP is a potent endogenous opioid that promotes appetite and has important effects in various types of pathological decreases in food intake [22].CCK-8 is a peptide hormone that is ubiquitous in both the gastrointestinal tract and brain,and has significant anorexic effects.Dysregulation in the ratio of the 2 hormones may be a cause of food intake decrease in animals.After juvenile rats were fed with special feed for 4 weeks,peripheral blood β-EP levels decreased and CCK-8 level increased.Administration of various doses of CPD JPXSF for four weeks increased β-EP expression and the 2.40 g/kg/d dose decreased CCK-8 expression.

Appetite-related gastrointestinal hormones can significantly affect the secretion,motility,and absorption functions of the feeding center and the gastrointestinal tract,bile duct,and pancreas,and has regulatory effects in the pathophysiology of anorexia.MTL is a polypeptide consisting of 22 amino acids and is mainly secreted by the endocrine cells in the duodenum-colon mucosa.MTL promotes stomach contractions and small intestine segmental motility.MTL levels are lower in anorexic children than in healthy children,resulting in insufficient gastric motility,poor gastric emptying,and increased gastric retention.This results in high gastric residue retention rates observed at 60,90,and 120 minutes after meals and leads to anorexia [23].GAS is a gastrointestinal hormone secreted by G cells in the gastric antrum and duodenum.Its main function is to stimulate gastric acid and pepsin secretion and accelerate intestinal peristalsis [24].After juvenile rats were fed with special feed for four weeks,peripheral blood MTL expression decreased and there was no significant change in GAS expression.However,administration of 2.40 g/kg/d CPD JPXSF for four weeks increased MTL expression,while there was no effect at any of the doses tested on GAS expression.

The results of this study showed that CPD JPXSF can increase MTL and stimulate pepsin secretions to accelerate gastric motility,increase gastrointestinal function,and promote food absorption.This thereby increases food intake and promotes weight gain.CPD JPXSF can also increase β-EP and inhibit CCK-8 to promote appetite and ameliorate anorexia symptoms.CPD JPXSF consists of traditional Chinese medicines that can be taken as both food and drug in addition to typical medicinal foods such as fungi,which can promote appetite and digestion and increase food intake and weight in anorexic juvenile rats.Therefore,it may promote digestion and improve appetite in adolescents with anorexia,and this finding provides a laboratory basis for clinical applications.However,there are many causes of pediatric anorexia,and other animal models are still required for efficacy evaluation.Lastly,clinical studies are required to prove the therapeutic effects of CPD JPXSF in patients with pediatric anorexia.