anti-arthritic effects of microneedling with bee venom gel · anti-arthritic effects of...

7
Anti-arthritic effects of microneedling with bee venom gel Mengdi Zhao a , Jie Bai a , Yang Lu*, Shouying Du*, Kexin Shang, Pengyue Li, Liu Yang, Boyu Dong, Ning Tan School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China Received 14 June 2016; accepted 30 August 2016 Available online 8 October 2016 KEYWORDS Arthritis; Bee venom; Gel; Melittin; Microneedle Abstract Objective: To combine with transdermal drug delivery using microneedle to simu- late the bee venom therapy to evaluate the permeation of bee venom gel. Methods: In this study, the sodium urate and LPS were used on rats and mice to construct the model. Bee venom gelemicroneedle combination effect on the model is to determine the role of microneedle gel permeation by observing inflammation factors. Results: Compared with the model group, the bee venom gelemicroneedle combination group can reduce the level of serum nitric oxide of the acute gouty inflammation model caused by sodium urate, and on LPS induced mouse model of acute inflammation effect and the micro. Conclusions: Bee venom can significantly suppress the occurrence of gouty arthritis inflamma- tion in rats and mice LPS inflammatory reaction. Choose the 750 mm microneedle with 10N force on skin about 3 minutes, bee venom can play the optimal role, and the anti-inflammatory effect is obvious. Microneedles can promote the percutaneous absorption of the active macromolecules bee venom gel. ª 2017 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by- nc-nd/4.0/). Introduction Arthritis is an inflammatory disease that affects the joints and surrounding tissues. The chronic pain associated with arthritis affects the quality of life of sufferers Pharma- ceutical agents, such as nonsteroidal anti-inflammatory drugs, may be used to relieve inflammation and other symptoms, but their long-term use can lead to side-effects, such as diabetes and hypertension. 1 Bee venom (BV) has been used in traditional Chinese medicine (TCM) to relieve pain and treat inflammatory * Corresponding authors. E-mail addresses: [email protected] (Y. Lu), dusy@bucm. edu.cn (S. Du). Peer review under responsibility of Beijing University of Chinese Medicine. a Both authors contributed equally to this work. http://dx.doi.org/10.1016/j.jtcms.2016.09.005 2095-7548/ª 2017 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Available online at www.sciencedirect.com ScienceDirect journal homepage: http://www.elsevier.com/locate/jtcms Journal of Traditional Chinese Medical Sciences (2016) 3, 256e262

Upload: others

Post on 24-Dec-2019

6 views

Category:

Documents


0 download

TRANSCRIPT

Page 1: Anti-arthritic effects of microneedling with bee venom gel · Anti-arthritic effects of microneedling with bee venom gel Mengdi Zhao a, Jie Bai a, Yang Lu *, Shouying Du , Kexin Shang,

Journal of Traditional Chinese Medical Sciences (2016) 3, 256e262

Available online at www.sciencedirect.com

ScienceDirect

journal homepage: http: / /www.elsevier .com/locate/ j tcms

Anti-arthritic effects of microneedling withbee venom gel

Mengdi Zhao a, Jie Bai a, Yang Lu*, Shouying Du*, Kexin Shang,Pengyue Li, Liu Yang, Boyu Dong, Ning Tan

School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China

Received 14 June 2016; accepted 30 August 2016Available online 8 October 2016

KEYWORDSArthritis;Bee venom;Gel;Melittin;Microneedle

* Corresponding authors.E-mail addresses: landocean28@1

edu.cn (S. Du).Peer review under responsibility of

Medicine.a Both authors contributed equally

http://dx.doi.org/10.1016/j.jtcms.202095-7548/ª 2017 Beijing University oCC BY-NC-ND license (http://creative

Abstract Objective: To combine with transdermal drug delivery using microneedle to simu-late the bee venom therapy to evaluate the permeation of bee venom gel.Methods: In this study, the sodium urate and LPS were used on rats and mice to construct themodel. Bee venom gelemicroneedle combination effect on the model is to determine the roleof microneedle gel permeation by observing inflammation factors.Results: Compared with the model group, the bee venom gelemicroneedle combination groupcan reduce the level of serum nitric oxide of the acute gouty inflammation model caused bysodium urate, and on LPS induced mouse model of acute inflammation effect and the micro.Conclusions: Bee venom can significantly suppress the occurrence of gouty arthritis inflamma-tion in rats and mice LPS inflammatory reaction. Choose the 750 mmmicroneedle with 10N forceon skin about 3minutes, bee venomcanplay the optimal role, and the anti-inflammatory effect isobvious. Microneedles can promote the percutaneous absorption of the active macromoleculesbee venom gel.ª 2017 Beijing University of Chinese Medicine. Production and hosting by Elsevier B.V. This is anopen access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

63.com (Y. Lu), dusy@bucm.

Beijing University of Chinese

to this work.

16.09.005f Chinese Medicine. Production acommons.org/licenses/by-nc-nd/

Introduction

Arthritis is an inflammatory disease that affects the jointsand surrounding tissues. The chronic pain associated witharthritis affects the quality of life of sufferers Pharma-ceutical agents, such as nonsteroidal anti-inflammatorydrugs, may be used to relieve inflammation and othersymptoms, but their long-term use can lead to side-effects,such as diabetes and hypertension.1

Bee venom (BV) has been used in traditional Chinesemedicine (TCM) to relieve pain and treat inflammatory

nd hosting by Elsevier B.V. This is an open access article under the4.0/).

Page 2: Anti-arthritic effects of microneedling with bee venom gel · Anti-arthritic effects of microneedling with bee venom gel Mengdi Zhao a, Jie Bai a, Yang Lu *, Shouying Du , Kexin Shang,

Anti-arthritic effects of microneedling with bee venom gel 257

diseases such as rheumatoid arthritis2 BV has anti-inflammatory, analgesic, and immune system-enhancingeffects.3

Bee venom is a fragrant light-yellow liquid secreted bystimulated worker bees.4 It is comprised of melittin,phospholipase A2, histamine, hyaluronidase, catechol-amine, and serotonin.5 Melittin is the main medicinalingredient, and accounts for 50% of the dry weight of BV.6

Melittin has strong anti-inflammatory and analgesic ef-fects, and can be combined into natural or artificialmembranes. Melittin has hormone-like effects, but nohormone-like side effects.7 It can activate release of ad-renocorticotropic hormone, reduce capillary permeability,and inhibit the synthesis of prostaglandin E2 and neutro-phils. It has been reported that melittin also can disruptthe nuclear factor-kappa B (NF-kB) pathway, inhibit the c-Jun N-terminal kinase pathway and hamper the activity ofNF-kB and signal transducer and activator of transcription-3.8e10 The analgesic intensity of melittin is 40% that ofmorphine and its analgesic duration is longer. BV does notaffect the digestive tract like salicylic acid-based drugsand it does not inhibit the immune system likecorticosteroids.11

In Chinese medicine, bee sting therapy is applied toacupoints to treat arthritis. This form of acupuncture pro-motes the flow of qi and blood, dredges the channels, andexpels pathogens, while simultaneously enabling BV toelicit its anti-inflammatory and analgesic effects.12e13

However, live bee stings can result in pain, itching, or anallergic reaction.14

Transdermal drug delivery (TDD) offers several advan-tages over systemic administration, such as oral and intra-venous.15 While the early TDD approach, the transdermalpatch, is advantageous, only small-molecule drugs can beabsorbed because of the barrier action of the skin’s stratumcorneum. The recent development of microneedles is anattempt to circumvent the stratum corneum barrier.16

Microneedles can be used to enhance TDD.17 Gel is a solidjelly-like material, it made by extract and matrix. Gelshave good biocompatibility, can extend the time for whichthe effect occurs, are associated with good patientcompliance, and are suitable for polypeptide drugs.18 Themirconeedle can breakdown the skin and make the thera-peutic dose gel into the skin.19

In the present study, a bee venom in gel form was pre-pared. Melittin is not stable in water and the main factoraffecting its stability is oxidation. We used the stability ofmelittin as an index to screen additives for gel preparation.We created two models in experimental animal models:acute gouty inflammation induced by sodium uratein rats-and acute inflammation induced by lipopolysaccharide(LPS) in mice. We then tested microneedling with beevenom gel (BVG) in these two models to verify the anti-inflammatory effect.

Materials and methods

Animals

Animal experiments were carried out according to theGuidelines for the Care and Use of Laboratory Animals

published by the Beijing University of Chinese Medicine(Beijing, China).

Male Kunming mice (18e22 g) and SpragueeDawley rats(SCXX 2006-0009)were purchased from Vital River Labora-tories (Beijing, China) and randomly allocated into 8 groupsof 8 animals each. Eight mice were housed in a cage atambient temperature and pressure.

Screening of bee venom gel materials and selectionof antioxidants

First, an appropriate gel matrix was chosen from CMC-Na,Methylcellulose, carbomer 934, Sodium alga acid, Hydrox-ypropyl methylcellulose-E15. Then, 0.2% vitamin C, 0.2%citric acid, 0.2% sodium sulfite, 0.2% thiourea, 0.2% sodiumthiosulfate, 0.2% glucose, 0.2% mannitol, 0.2% gelatin and0.1% stabilizer S. After 72 hours, the content and retentionrate of melittin was determined to select the suitableantioxidant. The concentration of the antioxidant wasdetermined by comparing the viscosity and calculating theretention rate of melittin in the 60�C heating test.

Bee venom gel preparation

Bee venom (Hangzhou Tianchumiyuan Health Products,Hangzhou, China) with a total protein content of approxi-mately 80% was weighed and mixed with deoxygenatedwater (Hangzhou Wahaha Group, Hangzhou, China) to pre-pare 100 mg/mL BV solution. To this was added the antiox-idant. The mixture was then dissolved in 10% propyleneglycol followed by addition of 0.01% butylparaben. Theresultant mixture was added to the matrix to form BVG.

Accelerated stability test

The appearance of the BVG was uniform and transparent,with a pH of 7.53. There was no discoloration, phase sep-aration, or peculiar smell in 6 months. Centrifugation at2500 rpm for 30 minutes at 25�C did not result in stratifi-cation. The BVG was placed in a constant climate chamber(LHS-100CL; Shanghai Scientific Instruments & Materials,Shanghai, China) for 6 months. Melittin content at 0, 1, 2,3, and 6 months was measured. The appearance, shape andconcentration of melittin were observed.

In vitro study of melittin release from BVG

Release of melittin from BVG with and without stabilizer wasstudied. Using a Franz-type diffusion cell, a microporousmembrane (0.8 mm) was placed between the diffusion andreceiving chambers. Volume of the receiving chamber was18 mL with a diffusion area of 3.14 cm2. Deaerated waterwas added to the receiving chamber until the liquid surfacewas fully in contact with the microporous membrane. Thetwo types of gels were placed on the microporous mem-brane, followed by magnetic stirring at 300 rpm for 72 hoursat 32�C.To draw receptor fluid (1.0 mL) at 0.5, 1, 2, 4, 6 12,24, 36, 48, 60, and 72 hours simultaneously, the same vol-ume of water was added the chamber. Melittin content wasdetermined by high-performance liquid chromatography.

Page 3: Anti-arthritic effects of microneedling with bee venom gel · Anti-arthritic effects of microneedling with bee venom gel Mengdi Zhao a, Jie Bai a, Yang Lu *, Shouying Du , Kexin Shang,

258 M. Zhao et al.

Different conditions of microneedles in rat skinin vitro

Five groups were created: A, no microneedle; B, 250-mmmicroneedle with 10-N force on the skin for 3 minutes; C,750-mm microneedle with 10-N force on the skin for30 seconds; D, 750-mm microneedle with 5-N force on theskin for 3 minutes; E, 750-mm microneedle with a 10-Nforce on the skin for 3 minutes.

The animals were applied 1000ı̀m DTR microneedles. Ratskin was fixed in 10% formalin and dehydrated in a gradedseries of alcohol. Skin was embedded in paraffin andstained with hematoxylin and eosin, followed by observa-tion under light microscopy.

Effect of microneedling plus BVG on sodium urate-induced acute gouty arthritis

Modeling and animal groupingMale SD rats were anesthetized with 10% chloral hydrate.After administration 0.5 hour, 5% sodium urate (0.2 mL) wasinjected into the joint cavity of the right hind ankle.

Rats were divided into eight groups of 8 animals each(Table 1).

Microneedling of ratsRats were anesthetized with 10% chloral hydrate. Fur onthe back of each rat was shaved 12 hours prior to micro-needling. Apart from the normal, model, and bee venom Igroups, microneedling was performed on the back of eachanimal using various parameters based on the group theanimal belonged to (Table 1). At 24 and 30 hours, the backof each animal was dressed.

Effect of microneedling plus BVG on sodium urate-induced level of nitric oxide in serum

Nitric oxide (NO) is a proinflammatory factor in arthritis thatis commonly elevated in serum. Forty-eight hours aftermodeling, the blood sample was drawn from abdominalaorta. Samples were centrifuged 5000 rpm for 20 minutes at25�C to prepare serum, which was stored at �80�C. Thecontent of NOwas determinate by enzymemark instrument.

Table 1 Animal groups and test conditions.

Groups Treatment (mg/kg) Microneedlin

Microneedlin

Normal Normal saline (1.5) No microneeModel Normal saline (1.5) No microneePositive Dexamethasone (1.8) 750 mmBee venom Bee venom (1.5) 750 mmBee venom I Bee venom (1.5) No microneeBee venom II Bee venom (1.5) 250 mmBee venom group III Bee venom (1.5) 750 mmBee venom group IV Bee venom (1.5) 750 mm

Effect of a microneedle plus BVG on LPS-inducedacute inflammation in mice

Modeling and animal groupingTo induce inflammation, LPS (4.8 mg/kg body weight) wasinjected into the abdominal cavities of male Kunming mice.Mice were divided into 8 groups of 10 mice each (Table 2).

Microneedling of miceAfter anesthetization with 10% chloral hydrate, an area ofskin 1 cm � 1 cm was excised from the back of each mouse12 hours prior to the experiment. Apart from the normal,model, and bee venom I groups, microneedling was per-formed on the back of each animal using various parame-ters based on the group the animal belonged to (Table 2).This was followed by application of normal saline, dexa-methasone ointment and BVG.

Effect of a microneedling plus BVG on LPS-inducedNO levels in serum

Two hours after modeling, blood samples were drawn fromthe eye orbit. Samples were centrifuged (1000 rpm for10 min at 25�C) to prepare serum, which was stored at�20�C. The content of NO was determinate by enzymemark instrument.

Results

Screening of BVG materials and selection ofantioxidants

The appropriate gel matrix was chosen, the matrix is CMC-Na, the appearance gel which used the CMC-Na is colorless,tasteless and translucent.

CMC-Na was used as the gel matrix. Therefore, ensuingexperiments were conducted for CMC-Na gel. We usedthiourea, sodium thiosulfate, glucose, and stabilizer S asantioxidants (Tables 3 and 4). The gel was stable after72 hours. Apart from thiourea and stabilizer S, gels withthe other additives were not stable at 7 days. In theglucose gel, glucose precipitated, and melittin degradedcompletely. Mannitol, gelatin, and glucose could combine

g parameters

g performed (needle size) Force (N) Duration (min)

dling e e

dling e e

10 3.010 3.0

dling 10 3.010 3.010 0.55 3.0

Page 4: Anti-arthritic effects of microneedling with bee venom gel · Anti-arthritic effects of microneedling with bee venom gel Mengdi Zhao a, Jie Bai a, Yang Lu *, Shouying Du , Kexin Shang,

Table 3 Screening of antioxidants by comparing the retention rate of melittin.

Antioxidants Melittin retention rateafter 72 hours, % (SD)

Viscosity (Pa S) Retention rate of melittinafter 7 days, % (SD)

0.2% vitamin C 25.41 (3.88) Undetected Undetected0.2% citrate 1.50 (0.74) Undetected Undetected0.2% sodium sulfite 56.71 (3.04) Undetected Undetected0.2% thiourea 99.26 (2.10) 0.244 101.68 (3.59)0.2% sodium thiosulfate 98.10 (1.28) 0.495 93.46 (1.76)0.2% glucose 99.82 (2.38) 0.20 Undetected0.2% mannitol 55.66 (0.91) Undetected Undetected0.2% gelatin 71.71 (0.80) Undetected Undetected0.1% stabilizer S 101.28 (3.74) 0.286 102.03 (0.46)

Table 2 Animal groups and test conditions.

Groups Treatment (mg/kg) Microneedling parameters

Microneedling performed (needle size) Force (N) Duration (min)

Normal Normal saline (1.5) No microneedling e e

Model Normal saline (1.5) No microneedling e e

Positive Dexamethasone (1.8) 750 mm 10 3.0Bee venom Bee venom (5) 750 mm 10 3.0Bee venom I Bee venom (5) No microneedling 10 3.0Bee venom II Bee venom (5) 250 mm 10 3.0Bee venom group III Bee venom (5) 750 mm 10 0.5Bee venom group IV Bee venom (1.5) 750 mm 5 3.0

Anti-arthritic effects of microneedling with bee venom gel 259

with melittin, form stable hydrogen bonds and a nativeconformation, which could enhance the stability of thegel, but this stability was temporary. Rupture of thestructure resulted in gradual precipitation of the additive.In the gel with thiourea and stabilizer S, melittin wasstable and gel color did not change. The gel with sodiumthiosulfate turned yellow after 5 days, and was notselected.

The 60�C heating test showed that melittin in the thio-urea gel was not stable, and that thiourea caused severeirritation people use the thiourea for a long time, it can beabsorbed through the skin and cause influence heart func-tion, palsy the CNS. Compared with 0.05% stabilizer S, 0.1%stabilizer S was more stable. Possibly, the 0.05% stabilizer S

Table 4 Screening of thiorea and stabilizer S concentra-tions by comparing their retention rates of melittin.

Concentration(%)

Gel viscosity(Pa S)

Melittin retentionrate after 12 hoursin 60�C, % (SD)

Thiourea 0.05 0.188 4.67 (0.72)0.2 0.244 10.79 (1.43)1.0 0.232 41.66 (0.37)5.0 0.203 64.27 (2.66)

Stabilizer S 0.05 0.223 38.47 (3.14)0.1 0.254 97.98 (0.67)0.2 0.286 98.57 (0.94)1.0 0.195 Undetected

could not reach the critical micelle concentration, whichaffected the stability and thus detection of melittin. Forthis reason, the 0.1% stabilizer S was chosen as the additivefor the BVG.

Accelerated stability study

Melittin in the gel was stable during the period of storage (6months) (Fig. 1). The gel was a colorless semi-solid trans-lucent substance. There was no discoloration, phase sepa-ration, or peculiar smell.

In vitro release

The cumulative release rate of melittin was determinedusing the formula shown below:

Figure 1 Accelerated stability test of melittin in bee venomgel over 6 months.

Page 5: Anti-arthritic effects of microneedling with bee venom gel · Anti-arthritic effects of microneedling with bee venom gel Mengdi Zhao a, Jie Bai a, Yang Lu *, Shouying Du , Kexin Shang,

260 M. Zhao et al.

Q%Z

VCn þ

Xn�1

iZ1

VSCi

!,W � 100

V: Volume of receptor fluid;VS: Volume of receptor fluid drawn;W: Melittin content in the gel;Cn: Concentration of melittin in receptor fluid at N hours.

Figure 2 Cumulative release rate of melittin from stabilizer S.

Figure 3 Skin channels produced by varying microneedle param10-N force on the skin for 3 minutes; C, 750-mm microneedle withwith 5-N force on the skin for 3 minutes; E, 750-mm microneedle w

Melittin in a gel without stabilizer S showed rapidrelease before 6 hours, after which release was moregradual (Fig. 2). After 48 hours, degradation rate of melittinwas faster than the release rate, and its concentrationdecreased gradually. Melittin in the normal gel did notrelease in the first 2 hours and showed a slow release rateat 2e6 hours. At 6 hours, melittin release was rapid. After24 hours, degradation rate of melittin was faster than therelease rate, and its concentration decreased gradually.

Effects of varying microneedle parameters on ratskin

Varying parameters applied during microneedling resultedin different depths and sizes of channels in rat skin(Fig. 3). Compared with group A, needling in group Bfailed to break down the cuticle of the skin, forming asuperficial channel. In groups C and D, needling lead tobreak down of the cuticle and the superficial channel.Needling in group E resulted in cuticle breakdown and adeep channel.

eters. Note: A, no microneedling; B, 250-mm microneedle with10-N force on the skin for 30 seconds; D, 750-mm microneedleith a 10-N force on the skin for 3 minutes.

Page 6: Anti-arthritic effects of microneedling with bee venom gel · Anti-arthritic effects of microneedling with bee venom gel Mengdi Zhao a, Jie Bai a, Yang Lu *, Shouying Du , Kexin Shang,

Anti-arthritic effects of microneedling with bee venom gel 261

Effect of microneedling plus BVG on sodium urate-induced serum levels of NO in rats

Compared with the normal and model groups, serum NOlevels in the BVG group rats were markedly reduced(P < .01) (Fig. 4), especially in animals that underwent 750-mm microneedling with 10-N force.

Effect of microneedling with BVG on LPS-inducedacute inflammation in mice

Compared with the normal saline LPS group and normalsaline, serum NO level was obviously reduced in the BVGmice (**P < .01) (Fig. 5). Serum NO levels did not differsubstantially in the normal saline LPS group, the groupwithout a microneedling, and the group that underwent750-mm microneedling with 5-N force for 3 minutes. How-ever, serum NO levels in other groups that underwent

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

NO

μm

ol/L

**** **

** **

****

Figure 4 Effects of varying microneedling parameters plusbee venom gel on serum NO of gouty arthritis.Note: **P < .01 compared with the model group.

0.00

2.00

4.00

6.00

8.00

10.00

12.00

14.00

16.00

NO/μmol/L

** ** **

**

*

Figure 5 Effects of varying microneedling parameters plusbee venom gel on LPS-induced acute inflammation. Note:*P < .05, compared with the LPS þ normal saline; **P < .01,compared with the model group.

microneedling with BVG using varying parameters werereduced, in particular animals in the 750 mm microneedlingwith 10 N force for 3 minutes.

Discussion

We used the stability of melittin as an indicator to screen aBVG formulation in rats and mice. We prepared a stableBVG and combined it with microneedling technology tostudy the penetration ability of BVG in the murine modelsof arthritis.

In the previous study, Melittin is not stability in water, atpreparation course, the instability would create waste theMelittin and affect the efficacy. By examining the stabilityof melittin in gels with different additives, we found thatmelittin in a gel with 0.1% stabilizer was stable for 6months.

Our results indicate the bee venom and its melittincontent could reduce NO levels in MUC- and LPS-inducedinflammation. Different penetration enhancements wereseen under different microneedling parameters. Based onthe skin experiments, melittin could not penetrate the skinwithout the assistance of the microneedle. Shorter micro-needles could not puncture the stratum corneum, thus ef-ficacy was not obvious. Moreover, the smaller force and theshorter application time, the lesser the amount of melittinpenetration, such that the therapeutic dose could not bereached. Our results indicated that a 750-mm microneedlewith a 10-N force applied for 3 minutes had the greatestanti-inflammatory effect.

Conclusion

Microneedles can promote percutaneous absorption ofactive macromolecules in bee venom gel. The present studyprovides an experimental basis for microneedleadministration.

Author contributions

MDZ, KXS, and JB conducted the experiments. SYD and YLdesigned the study and provided the reagents and mate-rials. MDZ undertook all the percutaneous experiment anddrafted the manuscript. PYL, LY, BYD, and NT performedthe statistical analyses, drafted and critically revisedthe manuscript. All authors read and approved themanuscript.

Funding

This study was supported by the Beijing Science andTechnology New Star Program (grant No. 2015A048), andthe Young Teacher Special of Beijing University of ChineseMedicine (1000061221025) and National Science and Tech-nology Major New Drug Projects (2014ZX09301306-009).

Page 7: Anti-arthritic effects of microneedling with bee venom gel · Anti-arthritic effects of microneedling with bee venom gel Mengdi Zhao a, Jie Bai a, Yang Lu *, Shouying Du , Kexin Shang,

262 M. Zhao et al.

References

1. Huang SS. Analysis of the treatment of rheumatoid arthritis.Chin J Pharm Econ. 2012;5:319e320.

2. Hui CL, Lin B, Zheng CJ, Qin LP, Han T. Research progress inNatural Medicine of anti-rheumatoid arthritis and its mecha-nism. Chin Tradit Herb Drugs. 2011;42(7):1435e1440.

3. Liu HY, Tong FD. The research on bee venom and its clinicalapplication. Chin Med Mater. 2003;26(6):456e458.

4. Chen SL. The Apicultural Science in China. Beijing: ChinaAgriculture Press; 2001:666.

5. Ferreira Junior RS, Sciani JM, Marques-Porto R, et al. African-ized honey bee (Apis mellifera) venom profiling: seasonalvariation of melittin and phospholipase A (2) levels. Toxicon.2010;56(3):355e362.

6. Li RL. Bee Venom Proteome Research [dissertation]. Beijing:Chinese Academy of Agricultural Sciences; 2013 [Chinese].

7. Yan XB. Bee venom injection in the treatment of rheumatic andrheumatoid arthritis 86 cases. Chin J Integr Tradit West Med.1995;15(6):370.

8. Kima SK, Park KY, Yoon WC. Melittin enhances apoptosisthrough suppression of IL-6/sIL-6R complex-induced NF-kB andSTAT3 activation and Bcl-2 expression for human fibroblast-likesynoviocytes in rheumatoid arthritis. Jt Bone Spine. 2011;78(5):471e477.

9. Yun SW, Bae GS, Kim MS. Melittin inhibits cerulein-inducedacute pancreatitis via inhibition of the JNK pathway. IntImmunopharmacol. 2011;11:2062e2072.

10. Park JH, Kum YS, Lee TI. Melittin attenuates liver injury inthioacetamide-treated mice through modulating

inflammation and fibrogenesis. Exp Biol Med. 2011;236(11):1306e1313.

11. Guan ZH. Clinical research of acupuncture point with beetherapy. J Clin Acupunct Moxib. 1998;14(6):32.

12. Gong Y, Wang JS, Liao XQ,Fang WF. Research status in thebee venom treat rheumatoid arthritis. Apic Chin. 2013;06:44e47.

13. Li X, Guo MQ. The mechanism of bee acupuncture and theapplication of bee venom in rheumatoid arthritis and oste-oarthritis. J Tradit Chin Orthop Traumatol. 2016;28(2):69e72.

14. Li WY, Zhao BB, Cui SS. Thinking and countermeasures of beeacupuncture therapy and safety research. Apic Chin. 2010;61(1):36e37.

15. Yang J, Hu J, He B, Cheng Y. Transdermal delivery of thera-peutic agents using dendrimers (US20140018435A1): a patentevaluation. Expert Opin Ther Pat. 2015;6:1e6.

16. Cahill EM, O’Cearbhaill ED. Toward biofunctional micro nee-dles for stimulus responsive drug delivery. Bioconjug Chem.2015;26:1289e1296.

17. Ita K. Transdermal delivery of drugs with microneedles-potential and challenges. Pharmaceutics. 2015;7(3):90e105.

18. Lai BL, Wang LS, Zhang S, Tian Y. The advance in Chinesemedicine gel. Tradit Chin Drug Res Clin Pharmacol. 2010;21(2):211e213.

19. Gomma YA, Morrow DI, Garland MJ, Donnelly RF, El-Khordagui LK, Meidan VM. Effects of microneedle length,density, insertion time and multiple applications on human skinbarrier function: assessments by transepidermal water loss.Toxicol Vitro. 2010;24:1971e1978.